CN101394864A - Compositions that include hemagglutinin, methods of making and methods of use thereof - Google Patents

Abstract

Methods of making compositions that stimulate a protective immune response in a subject include a portion of a protein from a naturally occurring viral hemagglutinin, wherein the portion includes at least a portion of a globular head, includes at least a portion of at least one secondary structure that causes the globular head to essentially retain its tertiary structure, and lacks a membrane fusion domain, a transmembrane domain and a cytoplasmic domain. Compositions comprise a flagellin component or a Toll-like Receptor agonist component that is at least a portion of a flagellin or a Toll-like Receptor agonist, wherein the flagellin component or Toll-like Receptor agonist component includes at least one cysteine residue and whereby the flagellin component or Toll-like Receptor agonist component activates a Toll-like Receptor 5 or Toll-like Receptor. Compositions comprise a flagellin component that is at least a portion of a flagellin, wherein at least one lysine of the flagellin component has been substituted with at least one arginine, serine and histidine, whereby the flagellin component activates Toll-like Receptor 5. Compositions can further include an antigen, such as an influenza antigen, a flavivirus antigen, a pathogen-related antigen, a bacterial capsular antigen and a carrier protein. The compositions are used to stimulate an immune response and a protective immune response in a subject.

Description

The compositions, the method for making it and the method for using it that comprise hemagglutinin

Related application

The application's case is a U.S. Provisional Application case the 60/779th, No. 854 (application on March 7th, 2006), the 60/784th, No. 497 (application on March 20th, 2006), the 60/790th, No. 457 (application on April 7th, 2006), the 60/814th, No. 292 (application on June 16th, 2006), the 60/830th, No. 881 (the continuity case of application on 2006 July 14, the 60/838th, No. 007 (application on August 16th, 2006) and the 60/856th, No. 451 (application on November 3rd, 2006) is also advocated its priority.Whole teachings of above-mentioned application case are incorporated this paper into way of reference.

Background technology

Influenza infection may cause disease.The strategy of the disease that correlates in order to prevention management and control and influenza infection can comprise with inactivation of viruses and medicine injection vaccine.Yet this type of strategy may need to spend the supply that money is kept needs, and therefore its supply is limited; May cause variable protective effect and not satisfied sx, so invalid in prevention or treatment discomfort and consequence (being death) in some case with the disease of influenza infection connection.Therefore, have and need research and development to be used to prevent and novelty, improvement and the effective Therapeutic Method of the treatment of diseases that management and control and influenza infection correlate.

Summary of the invention

The invention relates to compositions, for example stimulate the compositions of protective immunological reaction, and make the method for protein that can stimulate individual interior protective immunological reaction.

In an embodiment, the present invention is a kind of method of protein that stimulates individual interior protective immunological reaction of making, it comprises isolates a proteinic part from the natural viral hemagglutinin, wherein this protein portion comprises at least a ball head, and a kind of at least a portion that makes ball head in fact still possess the secondary structure of its tertiary structure, and wherein this protein portion lacks film fusion function territory, strides film functional domain and kytoplasm functional domain; The nucleotide sequence of this protein portion of coding is converted in the prokaryotic host cell; Reach the proteinic step of cultivating this prokaryotic host cell and making the individual interior protective immunological reaction of stimulation thus.

In another embodiment, the present invention is a kind of method of protein that stimulates individual interior protective immunological reaction of making, it comprises isolates a proteinic part from the natural viral hemagglutinin, form protein portion thus, wherein this protein portion comprises at least a portion of ball head at least, and at least a at least a portion that makes ball head in fact still possess the secondary structure of its tertiary structure, and wherein this protein portion lacks film fusion function territory, strides film functional domain and kytoplasm functional domain; The nucleotide sequence commentaries on classics of this protein portion of coding is infected to the eucaryon host cell, and wherein this eukaryotic host cell is not pichia pastoris phaff (Pichia pastoris) eukaryotic host cell; Reach the proteinic step of cultivating this eukaryotic host cell and making the individual interior protective immunological reaction of stimulation thus.

In another embodiment, the present invention is a kind of method of protein that stimulates individual interior protective immunological reaction of making, it comprises isolates a proteinic part from the natural viral hemagglutinin, form protein portion thus, wherein this protein portion comprises at least a portion of ball head at least, and at least a at least a portion that makes ball head in fact still possess the secondary structure of its tertiary structure, and wherein this protein portion lacks film fusion function territory, strides film functional domain and kytoplasm functional domain; The nucleotide sequence commentaries on classics of this protein portion of coding is infected to the eucaryon host cell, and wherein this eukaryotic host cell is not Drosophila melanogaster (Drosophila melanogaster) eukaryotic host cell; Reach the proteinic step of cultivating this eukaryotic host cell and making the individual interior protective immunological reaction of stimulation thus.

In an embodiment, the present invention is a kind of method of protein that stimulates individual interior protective immunological reaction of making, it comprises isolates a proteinic part from the natural viral hemagglutinin, form protein portion thus, wherein this protein portion comprises at least a portion of ball head, and at least a at least a portion that makes ball head in fact still possess the secondary structure of its tertiary structure, and wherein this protein portion lacks film fusion function territory, strides film functional domain and kytoplasm functional domain; The nucleotide sequence commentaries on classics of this protein portion of coding is infected to the eucaryon host cell, and wherein this eukaryotic host cell is not the insecticide eukaryotic host cell; Reach the proteinic step of cultivating this eukaryotic host cell and making the individual interior protective immunological reaction of stimulation thus.

In an embodiment, the present invention is a kind of method of protein that stimulates individual interior protective immunological reaction of making, it comprises isolates a proteinic part from the natural viral hemagglutinin, form protein portion thus, wherein this protein portion comprises at least a portion of ball head, and at least a at least a portion that makes ball head in fact still possess the secondary structure of its tertiary structure, and wherein this protein portion lacks film fusion function territory, strides film functional domain and kytoplasm functional domain; The nucleotide sequence commentaries on classics of this protein portion of coding is infected to the eucaryon host cell, and wherein this eukaryotic host cell is not the insect host cell through stable conversion; Reach the proteinic step of cultivating this eukaryotic host cell and making the individual interior protective immunological reaction of stimulation thus.

In another embodiment, the present invention is a kind of method of protein that stimulates individual interior protective immunological reaction of making, it comprises isolates a proteinic part from the natural viral hemagglutinin, form protein portion thus, wherein this protein portion comprises at least a portion of ball head, and at least a at least a portion that makes ball head in fact still possess the secondary structure of its tertiary structure, and wherein this protein portion lacks film fusion function territory, strides film functional domain and kytoplasm functional domain; The nucleotide sequence commentaries on classics of this protein portion of coding is infected to the eucaryon host cell, and wherein this eukaryotic host cell is not the pichia pastoris phaff eukaryotic host cell, also is not through the stable insect host cell that infects that changes; Reach the proteinic step of cultivating this eukaryotic host cell and making the individual interior protective immunological reaction of stimulation thus.

In another embodiment, the present invention is a kind of method that stimulates individual interior protective immunological reaction, it comprises and contains the proteinic compositions administration that makes by the method that comprises following steps and give this individual step a kind of: a proteinic part is isolated from the natural viral hemagglutinin, form protein portion thus, wherein this protein portion comprises at least a portion of ball head, and at least a at least a portion that makes ball head in fact still possess the secondary structure of its tertiary structure, and wherein this protein portion lacks film fusion function territory, stride film functional domain and kytoplasm functional domain; The nucleotide sequence of this part of coding is converted in the prokaryotic host cell; Reach the protein of cultivating this prokaryotic host cell and making the individual interior protective immunological reaction of stimulation thus.

In other embodiment; the present invention is a kind of method that stimulates individual interior protective immunity; it comprises and comprises that with a kind of the compositions administration of the protein portion that derives from the natural viral hemagglutinin gives this individual step; wherein this protein portion comprises at least a portion of ball head; and a kind of at least a portion that makes ball head in fact still possess the secondary structure of its tertiary structure, and wherein this protein portion lacks film fusion function territory, strides film functional domain and kytoplasm functional domain.

In another embodiment, the present invention is a kind of method of making the viral hemagglutination fibroin that stimulates individual interior protective immunological reaction, it comprises isolates a proteinic part from the natural viral hemagglutinin, form protein portion thus, wherein this protein portion comprises at least a portion of ball head, and at least a at least a portion that makes ball head in fact still possess the secondary structure of its tertiary structure, and wherein this protein portion lacks film fusion function territory, strides film functional domain and kytoplasm functional domain; The nucleotide sequence commentaries on classics of this protein portion of coding is infected to the eucaryon host cell, and wherein this eukaryotic host cell is not the pichia pastoris phaff eukaryotic host cell, also is not through the stable insect host cell that infects that changes; Reach the proteinic step of cultivating this eukaryotic host cell and making the individual interior protective immunological reaction of this stimulation thus.

In another embodiment, the present invention is a kind of method of protein that stimulates individual interior protective immunological reaction of making, it comprises isolates a proteinic part from the natural viral hemagglutinin, form protein portion thus, wherein this protein portion comprises at least a portion of ball head, and at least a at least a portion that makes ball head in fact still possess the secondary structure of its tertiary structure, and wherein this protein portion lacks film fusion function territory, strides film functional domain and kytoplasm functional domain; The nucleotide sequence of this protein portion of coding is infected in the insect cell host cell; Reach the proteinic step of cultivating this insect host cell and making the individual interior protective immunological reaction of this stimulation thus.

In another embodiment, the present invention is a kind of method of making the viral hemagglutination fibroin that stimulates individual interior protective immunological reaction, it comprises the nucleotide sequence of at least a shortage of coding being striden the viral hemagglutination element of film functional domain and kytoplasm functional domain, transforms prokaryotic host cell; And cultivate this prokaryotic host cell, and make this proteinic step thus.

In other embodiment, the present invention is a kind of method that stimulates individual interior protective immunity, it comprises the proteinic compositions administration that makes by the method that may further comprise the steps and gives this individual step: comprised the nucleotide sequence of at least a shortage film of coding being striden the viral hemagglutination element of film functional domain and kytoplasm functional domain a kind of comprising by it, transform prokaryotic host cell; And this prokaryotic host cell of cultivation.

In another embodiment; the present invention is a kind of method that stimulates individual interior protective immunity; it comprises and comprises that with a kind of having at least a shortage strides the proteinic compositions administration of the viral hemagglutination element of film functional domain and kytoplasm functional domain and give this individual step, and wherein this protein expression is in prokaryotic host cell.

In other embodiment, the present invention is the compositions of the part of at least a portion of a kind of molecule pattern (pathogen-associated molecular pattern) that comprises at least a pathogen-connection and natural viral hemagglutinin, wherein the part of this natural viral hemagglutinin comprises at least a portion of ball head, and at least a at least a portion that makes ball head in fact still possess the secondary structure of its tertiary structure, and wherein the part of this natural viral hemagglutinin lacks film fusion function territory, stride film functional domain and kytoplasm functional domain.

Another embodiment of the present invention is a kind of compositions that comprises it for the flagellin composition of at least a portion of flagellin, and wherein this flagellin composition comprises at least one cysteine residues, and makes this flagellin composition activation clock sample receptor 5 thus.

In another embodiment, the present invention is a kind of compositions that comprises it for the clock sample receptor stimulating agent composition of at least a portion of clock sample receptor stimulating agent, wherein this clock sample receptor stimulating agent is formed and is comprised at least one cysteine residues, it is arranged in natural clock sample receptor stimulating agent and locating of cysteine residues do not occur, and makes this clock sample receptor stimulating agent form activation clock sample receptor thus.

In other embodiment, the present invention is a kind of compositions that comprises it for the flagellin composition of at least a portion of flagellin, wherein at least one lysine of this flagellin composition is replaced by at least one arginine, makes flagellin composition activation clock sample receptor 5 thus.

In another embodiment, the present invention is a kind of compositions that comprises it for the flagellin composition of at least a portion of flagellin, wherein at least one lysine of this flagellin composition is replaced by at least one serine residue, makes flagellin composition activation clock sample receptor 5 thus.

Another embodiment of the present invention, be a kind of compositions that comprises it for the flagellin composition of at least a portion of flagellin, wherein at least one lysine of this flagellin composition is replaced by at least one histidine residues, makes flagellin composition activation clock sample receptor 5 thus.

Other embodiment of the present invention; be a kind of method that stimulates individual interior protective immunity; it comprises this individual step is given in a kind of compositions administration of flagellin composition of at least a portion that is included as flagellin; wherein this flagellin composition comprises at least one cysteine residues, and makes flagellin composition activation clock sample receptor 5 thus.

In another embodiment; the present invention is a kind of method that stimulates individual interior protective immunity; it comprises this individual step is given in a kind of compositions administration of flagellin composition of at least a portion that is included as flagellin; wherein at least one lysine of this flagellin composition is replaced by at least one arginine, makes flagellin composition activation clock sample receptor 5 thus.

Other embodiment of the present invention; be a kind of method that stimulates individual interior protective immunity; it comprises this individual step is given in a kind of compositions administration of flagellin composition of at least a portion that is included as flagellin; wherein at least one lysine of this flagellin composition is replaced by at least one serine residue, makes flagellin composition activation clock sample receptor 5 thus.

In another embodiment; the present invention is a kind of method that stimulates individual interior protective immunity; it comprises this individual step is given in a kind of compositions administration of flagellin composition of at least a portion that is included as flagellin; wherein at least one lysine of this flagellin composition is replaced by at least one histidine residues, makes flagellin composition activation clock sample receptor 5 thus.

In another embodiment; the present invention is a kind of method that stimulates individual interior protective immunity; it comprises the compositions administration that a kind of clock sample receptor stimulating agent that is included as at least a portion of clock sample receptor stimulating agent is formed and gives this individual step; wherein this clock sample receptor stimulating agent is formed and is comprised at least one cysteine residues; it is arranged in natural clock sample receptor stimulating agent and locating of cysteine residues do not occur, and makes this clock sample receptor stimulating agent form activation clock sample receptor thus.

Method and composition of the present invention can be used for stimulating individual interior immunoreation, especially protective immunity.Advantage of the present invention comprises (for example) cost-efficient method and can be relative the compositions of relatively large production, for the disease that is used to prevent and treatment is relevant with influenza infection.Therefore claimed method and compositions can be used for prevention or treatment influenza infection, and can avoid the serious discomfort and the death that cause because of influenza infection.

Description of drawings

Fig. 1 describes the strip-chart of PR8A type influenza virus haemagglutinin (HA) crystal structure (1RU7).To scheme expression is HA trimer, monomer, ball head functional domain and three kinds of vaccine candidate persons' selection from left to right.The molecule or the functional domain of institute's interest are irised out with dotted line and to be emphasized, for example monomer in the trimer structure, and the ball head functional domain in monomer structure.Three kinds of vaccine candidate persons' functional domain border is to intersect the mark sign.Also each residue number of selecting of labelling in indivedual vaccine sketch maps.

Fig. 2 describes the hydrophobicity mapping analysis of Lee B//40HA (SEQ ID NO:36), use ProtScale (http://ca.expasy.org/tools → Primary structure analysis→ ProtScale) to determine that selected border is this proteinic hydrophilic region.

Fig. 3 describes the proteinic sandwich ELISA analysis of STF2.HA B strain.Elisa plate is coated with the antibody at flagellin, and cultivates specified protein to catch.Use resists-ferret antibody detection protein matter with the goat of demarcating through enzyme at the ferret antiserum of B/ Malaysia/2506/2004.

Fig. 4 describes the proteinic TLR biological activity of STF2.HA B strain.HEK293 (TLR5+) cell and specified protein cultivated spend the night, and collect cell culture supernatant liquid, and by elisa assay IL-8.

Fig. 5 describes the proteinic TLR biological activity of STF2.HA B strain.HEK293 (TLR5+) cell and specified protein cultivated spend the night, and collect cell culture supernatant liquid, and by elisa assay IL-8.

Fig. 6 is described in the BALB/c mouse dosage-dependency antibody response to STF2.HA1-2 (PR8) (SEQ ID NO:90).Mice (10/ group) is carried out immunity according to being instructed in the 0th and 14 day, and took a blood sample in the 12nd and 21 day.Resisted-HA IgG reaction by the ELISA check in the 28th day.Include convalescent antiserum (x) in as positive controls.Meansigma methods ± the SD of every group of 10 other serum of data representation.

Fig. 7 describes in vitro and serum reactivity through the influenza infection cell.The 21st day serum sample described in Fig. 6 cultivated with the mdck cell of imitating-reach the PR/8/34-infection.The OD of data represented 10 other serum/groups ₄₅₀Meansigma methods ± SD.

Fig. 8 A describes the survival rate that causes the BALB/c mouse of exempting from through STF2.HA1-2 (PR8) (SEQ ID NO:90).To derive from the mice of Fig. 8 and Fig. 9 with LD in the 28th day ₉₀(8x10 ³EID) A type influenza virus PR/8/34, intranasal administration excite (after exciting the 0th day).

Fig. 8 B describes the weight that causes the BALB/c mouse of exempting from through STF2.HA1-2 (PR8) (SEQ ID NO:90).To derive from the mice of Fig. 6 and Fig. 7 with LD in the 28th day ₉₀(8 x 10 ³EID) A type influenza virus PR/8/34, intranasal administration excite (after exciting the 0th day).This figure reflects based on measuring every group of average weight that individual animal reaches 19 days every day.

Fig. 8 C describes the clinical scores that causes the BALB/c mouse of exempting from through STF2.HA1-2 (PR8) (SEQ ID NO:90).To derive from the mice of Fig. 6 and Fig. 7 with LD in the 28th day ₉₀(8 x 10 ³EID) A type influenza virus PR/8/34, intranasal administration excite (after exciting the 0th day).This figure reflects based on measuring every group of average clinical scores that individual animal reaches 19 days every day.Being analyzed as follows of clinical scores: 4 points=health, 3 points=minimizing combing, 2 points=health is active to be lowered, and 1 point=dying.

Fig. 9 describe be obtained from the 0th and 14 day the STF2.HA1-2 with specified dosage (PR8) (SEQ IDNO:90) carry out immunity, and the neutralization activity of the immune serum of the BALB/c mouse of taking a blood sample in the 21st day.It is to be calculated as that the terminal point of each sample is tired, and suppresses to reach at least 50% tire by virus-mediated cytolysis.Calculate the geometrical mean ± SD and the drawing of each dosage group.

Figure 10 A and 10B are described in the 0th and 14 day with subcutaneous injection 3 μ g STF2.HA1-2 (PR8) (SEQID NO:90), or cause the anti--HA antibody response of the BALB/c mouse of exempting from 3 or 0.3 μ g STF2.HA1-2 (NV) (SEQ ID NO:95).In the 21st day, serum separated and check its reactivity by ELISA with HA and (Figure 10 B) reorganization flagellin (STF2) (SEQ ID NO:96) that (Figure 10 A) purification that (derives from BEI resource Cat#NR-660) from A type influenza virus/Vietnam/1203/2004 gets.

Figure 11 describes and uses Bac-to-Bac baculovirus (Baculovirus) expression system, to express the required flow chart of steps of pFastBac construct.

Figure 12 A and 12B are described in the anti--HA specific IgG that causes in the BALB/c mouse of exempting from through the STF2.HA1-1 fused protein and react.Mice (10/ group) caused with specified STF2.HA1-1 fused protein in the 0th and 14 day exempt from, and took a blood sample in the 21st day.Resist-HA and anti--flagellin IgG reaction by the ELISA check.Include convalescent antiserum in as positive controls.Meansigma methods ± the SD of every group of 10 other serum of data representation.

Figure 13 A describes the survival rate that causes the BALB/c mouse of exempting from through reorganization STF2.HA1-1 protein.In the 28th day, with the mice among Figure 12 through intranasal (i.n.) with LD ₉₀(8 x 10 ³EID) A type influenza virus PR/8/34 excites.Detect the survival rate 21 days of indivedual mices in exciting the back.

Figure 13 B describes the weight that causes the BALB/c mouse of exempting from through reorganization STF2.HA1-1 protein.In the 28th day, with the mice among Figure 12 through intranasal with LD ₉₀(8 x 10 ³EID) A type influenza virus PR/8/34 excites.This figure reflects based on measuring every group of average weight that individual animal reaches 19 days every day.

Figure 13 C describes the clinical analysis that causes the BALB/c mouse of exempting from through reorganization STF2.HA1-1 protein.In the 28th day, with the mice among Figure 12 through intranasal with LD ₉₀(8 x 10 ³EID) A type influenza virus PR/8/34 excites.This figure reflects based on measuring every group of average weight that individual animal reaches 21 days every day.Being analyzed as follows of clinical scores: 4 points=health, 3 points=minimizing combing, 2 points=health is active to be lowered, and 1 point=dying.

Figure 14 describes the anti--HA antibody titer that causes the mice after exempting from through 10 μ g STF2.HA1-2 (VN).

Figure 15 describes the anti--HA antibody titer that causes the mice after exempting from through 3 μ g STF2.HA1-2 (VN).

Figure 16 describes the anti--HA antibody titer that causes the mice after exempting from through 1 μ g STF2.HA1-2 (VN).

Figure 17 describes through 10 LD ₉₀The survival rate of the mice of A type influenza virus/Vietnam/1203/04 after exciting.

Figure 18 describes the aminoacid sequence of the total length flagellin (STF2) of wherein emphasizing out critical function territory and residue.Lycoperdon polymorphum Vitt=through testing definite TLR5 binding site (people such as Smith, 2003); The K=lysine residue; Underscore=corresponding to the flagellin sequence (the peptide connexon is not shown) of STF2 Δ construct.

Figure 19 describes the space-filling model of flagellin (STF2), and wherein TLR5 binding site (left side) is emphasized with Lycoperdon polymorphum Vitt with lysine residue (right-hand part).

Figure 20 describes the space-filling model of flagellin (STF2), and wherein the residue that still remaines in the STF2 Δ is emphasized with black.

Figure 21 describes the two step PCR clone's of pMT/STF2 Δ sketch map.

Figure 22 is described in the proteinic flagellin epitope of identification STF Δ among the ELISA.

Figure 23 describes the TLR5-dependency IL-8 secretion of HEK293 cell behind specified STF2 Δ protein boost.

Figure 24 describes the STF2 Δ. the S200 chromatographic analysis collection of illustrative plates of hinge Cys:CysH1C1.Space=tubing string voidage; Conjugate=protein: the eluting peak of peptide conjugates; Indicate A260, A280 and electric conductivity among the figure.

Figure 25 describes RAWhTLR5 cell (solid mark) or TLR5-negative RA W cell (hollow mark) through the STF2 Δ. hinge Cys (square), STF2 Δ. and hinge Cys:CysH1C1 conjugate (circle) or the post-stimulatory TLR5-dependency of STF2.OVA (triangle) IL-8 secretion.

Figure 26 describes the S200 chromatographic analysis collection of illustrative plates of STF2.4xH1C1.Space=tubing string voidage; Conjugate=protein: the eluting peak of peptide conjugates; Indicate A260, A280 and electric conductivity among the figure.

Figure 27 is described in through natural H1C1 peptide or Pam3Cys.H1C1 peptide and causes resisting-the H1C1 antibody response in the BALB/c mouse of exempting from.

Figure 28 is described in to cause through natural H1C1 peptide or Pam3Cys.H1C1 peptide and exempts from, then with LD ₉₀(8 x 10 ³The survival figure of the BALB/c mouse that A type influenza virus/Puerto Rico/8/34 virus excites EID).

Figure 29 describes the aminoacid sequence (SEQ ID NO:498) of salmonella typhimurium flagellin the 2nd type (fljB/STF2), and wherein hinge region underlines sign.

Figure 30 describes the nucleotide sequence (SEQ ID NO:499) of coding SEQ ID NO:498.The nucleotide sequence of coding hinge region underlines sign.

Figure 31 describes the aminoacid sequence (SEQ ID NO:500) of the fljB/STF2 (also being called " fljB/STF2 Δ " or " STF2 Δ " in this paper) that does not contain hinge region.

Figure 32 describes the nucleotide sequence (SEQ ID NO:501) of coding SEQ ID NO:500.

Figure 33 describes the coli flagellum albumen fliC aminoacid sequence of (also being called " escherichia coli fliC " in this paper) (SEQ ID NO:502), and wherein hinge region underlines sign.

Figure 34 describes the nucleotide sequence (SEQ ID NO:503) of coding SEQ ID NO:502.The nucleotide sequence of coding hinge region underlines sign.

Figure 35 describes the aminoacid sequence (SEQ ID NO:504) of Munich Salmonella flagellin fliC (also being called " Munich Salmonella fliC " in this paper), and wherein hinge region underlines sign.

Figure 36 describes the nucleotide sequence (SEQ ID NO:505) of coding SEQ ID NO:504.The nucleotide sequence of coding hinge region underlines sign.

Figure 37 describes the aminoacid sequence of pMT/STF2.The peptide connexon underlines sign, and the Bip secretion signal is runic (SEQ ID NO:506).

Figure 38 describes the nucleotide sequence (SEQ ID NO:507) of coding SEQ ID NO:506.The nucleotide sequence of encoded peptide connexon underlines sign, and coding Bip sequencing nucleic acid sequence is a runic.

Figure 39 describes the Pam3Cys.M2e fused protein.The aminoacid sequence of M2e (SEQ ID NO:510) is represented with the runic pattern.

Figure 40 describes the activation that antigen-represent sexual cell (APC) is transmitted by class-clock sample receptor (TLR) signal.

Figure 41 A and 41B describe the plasmid construction body of M2 (for example, SEQ ID NOS:510, the 554) amino-end that is used to express H1 and H5 (SEQ ID NO:536) A type influenza virus separated strain.PMT: based on the expression vector of metallothionein promoter.Bip: the secretory signal sequence of immunoglobulin binding proteins.STF2: the total length flagellin of salmonella typhimurium.STF2 Δ: the STF2 of hinge region disappearance.MCS: multiple cloning site.

Figure 42 describes through being designed for the plasmid construction body of the HA that expresses H1 and H5A type influenza virus separated strain.AOX1:pPICZ alpha expression carrier (Yin Weituo King Company, Ka Sibeide, AOX1 promoter CA).α f: saccharomycetic secretory signal sequence.STF2: the total length flagellin of salmonella typhimurium.STF2 Δ: the STF2 of hinge region disappearance.MCS: multiple cloning site.

Figure 43 describes the aminoacid sequence (SEQ ID NO:561) of HA (PR8).

Figure 44 describes the nucleotide sequence (SEQ ID NO:562) of coding SEQ ID NO:561.

Figure 45 describes the aminoacid sequence (SEQ ID NO:563) of the large intestine bar fliC that does not contain hinge region.

Figure 46 describes the aminoacid sequence of pMT/STF2 Δ (SEQ ID NO:585).The peptide connexon underlines sign, and the Bip secretion signal is a runic.

Figure 47 describes the nucleotide sequence (SEQ ID NO:586) of coding SEQ ID NO:585.The nucleotide sequence of encoded peptide connexon underlines sign, and coding Bip sequencing nucleic acid sequence is a runic.

Figure 48 describes the aminoacid sequence (SEQ ID NO:595) of Munich Salmonella fliC (it also is called " Munich Salmonella fliC Δ " in this paper) that does not contain hinge region.

Figure 49 describes Munich Salmonella fliC Δ nucleotide sequence (SEQID NO:596) of coding SEQ ID NO:595.

Figure 50 describes the TLR5+ cell and secretes through post-stimulatory IL-8.

Figure 51 describes the TLR2+ cell and secretes through post-stimulatory TNF.

Figure 52 describes the M2e-specific IgG.

Figure 53 describes the OVA-specific IgG.

Figure 54 describes M2e-specific IgG serum titer.

The M2e-specific serum IgG that Figure 55 describes after short the liter tires.

Figure 56 describes the Pam3Cys.M2e dose response.

Figure 57 describes M2e-specific serum IgG and tires.

Figure 58 describes the rabbit igg reaction to M2e.

Figure 59 is described in the immunogenicity that causes STF2.4xM2e in the back 14 days rabbit.

Figure 60 describes the survival rate of the BALB/c mouse after exciting.Mice was exempted from according to indicated causing in illustrating in the 0th and 14 day, and passed through intranasal administration LD in the 28th day ₉₀A type influenza virus/Puerto Rico/8/34 virus excite (after exciting the 0th day).Reach 21 days in the survival rate that excites the back to detect mice.

Figure 61 describes the fusion constructs in the pET24 carrier.The T7:T7 promoter; The lacO:lac operon; STF2: salmonella typhimurium flagellin; The STF2 that the STF2 Δ=its hinge region has lacked; EIII ⁺Be the functional domain III of west nile virus envelope protein, it has amino acid whose 6 aminoacid of functional domain I.

Figure 62 A and 62B describe the TLR5 biological activity of STF2.EIII+ (SEQ ID NOS:657,658) and STF2 Δ EIII+ (SEQ ID NOS:673,674) fusion rotein.Purified proteic serial dilution is added to HEK293 (TLR5+) cell spend the night, and measure IL-8 content by ELISA.Use purified STF2.OVA as positive controls (Figure 129 A).Use TLR2 antagonist Pam3CSK4 as negative control group (Figure 129 B).

Figure 63 describes the STF2 Δ .EIII+ epitope that gets by elisa assay.Plate coated with total length WNE (open tubular column) (SEQ ID NO:642) or STF2 Δ .EIII+ (SEQ ID NOS:673,674), and is surveyed with specified antibody (mAb).Poly=is at the polyclonal antiserum of WNE; 3D9 to 7H2=is to the neutralizing monoclonal antibody of WNE epitope; Anti--flagellin=at the monoclonal antibody of flagellin.

Figure 64 A, Figure 64 B, Figure 64 C and Figure 64 D describe STF2.E (SEQ ID NOS:761,762); STF2.EIII+ (SEQ ID NOS:657,658) and STF2 Δ .EIII+ (SEQ ID NOS:673,674) fusion rotein and reactivity at the monoclonal antibody of WNE and flagellin.With plate coated with fusion rotein, blockade and with cultivate at the antibody of WNE or flagellin.Cultivate the back in species specificity IgG and detect antibody response with the HRP-demarcation.There is colour generation down in plate in tmb substrate, and machine read by use TECAN plate meter and Magellian software reads O.D.450/650.

Figure 65 describes with the IgG serum after the fusion rotein injection.With mice with PBS, contain the fruit bat conditioned medium (CM of STF2.E, positive controls), STF2 Δ .EIII+ (SEQ IDNOS:673, the 674) i.p. of 25 μ g, 25 μ g STF2 Δ .EIII+s.c., 25 μ g STF2.EIII+ (SEQ IDNO:659,658) i.p., 25 μ g STF2.EIII+ (SEQ ID NOS:657,658) or 25 μ g STF2.E (SEQID NOS:761,762) cause and exempt from.In the 35th day, will excite with WNV through causing the animal of exempting from.The serum that will derive from indivedual mices (the 35th day) is by direct ELISA characterization, and measure IgG concentration.Be to use purified WNV-E albumen (SEQ ID NO:642) as antigen in this analysis.This antigen (60) is to make in fruit bat, is a kind of protein of the his-of adding label.

Figure 66 describes the protection immunity that STF2 Δ .EIII+ (SEQ ID NOS:673674) and STF2.EIII+ (SEQ ID NOS:657,658) excite WNV virus.Mice caused exempt from, and excite in the 35th day WNV Strain 2741 with fatal dose.Detect survival rate and reach 21 days.

Figure 67 describes through fusion rotein and causes IgG serum titer after exempting from.STF2 Δ .EIII+ albumen brings out the WNV-specific IgG antibodies.With mice in the 0th, 14 and 28 day STF2 Δ .EIII+ with independent PBS or about 25 μ g (SEQ ID NOS:673,674) (045[positive controls]), STF2 Δ .EIII+ (067, trimer), STF2 Δ .EIII+ (070, monomer) or STF2 Δ .EIIIs+ (SEQ ID NOS:675,676) (069) cause and exempt from.In the 35th day, with the serum that derives from indivedual mices by direct ELISA characterization, and measure IgG concentration.Be to use purified WNV-E albumen (060, in fruit bat, make, be a kind of protein of the his-of adding label) as antigen in this analysis.

Figure 68 describes STF2 Δ .EIII+ in the mice (SEQ ID NOS:673,674) and STF2.EIII+ (SEQID NOS:657,658) avoids the protection immunity that WNV causes death and excites.In causing with fusion rotein after exempting from the 28th day all excites all groups with the WNV Strain 2741 of fatal dose, and detects survival rate and reach 21 days.The survival rate of each group (10 mice/group) is to represent with percentage ratio.

Figure 69 describes competition analysis.Must hang oneself causes serial dilution (five times of dilutions the start from 1:25) antiserum of exempting from mice and biotinylation WNE albumen (SEQ ID NO:642) is cultivated, and adds to then through each hole coated with the mAb 7H2 of the about 2mg/ml of concentration.Use Avidin-HRP with each hole colour generation, with measure because of with mAb 7H2 compete caused to the protein bound inhibition of West Nile.

Figure 70 describes the epitope mapping by STF2 Δ .EIII+ (SEQ ID NOS:675, the 676) antibody response that fused protein brought out.(E2-21, E27-E52 Figure 142) cause the immune serum of the animal of exempting from, check the ability of its identification corresponding to the overlapping peptide of the junction of the functional domain I of WNE envelope protein and III to the specified STF2 Δ-fused protein of must hanging oneself.

Figure 71 describes the epitope mapping of the antibody response that is brought out by STF Δ .EIIIs+ (SEQ ID NOS:675,676) E-21 (envelope protein) epitope fused protein.Assess hang oneself specified STF2 Δ-fused protein (E2-21, E2-21-1 (S, C), E2-21-2 (C, S), E2-21-2 (C, S) and E2-21-4 to E2-21-24, referring to Figure 139) cause the immune serum of the animal of exempting from, with the residue of the E-21 epitope that identifies definition west nile virus envelope protein.The data reflection replaces (with C, S represents) to its cysteine through serine; And follow-up seroreaction of carrying out the E-21 of aminoacid replacement with alanine.The peptide of tested person is shown in Figure 139.

Figure 72 describes the Pam3Cys.WNV001 (SEQ ID NO:771) that brings out the EIII-specific IgG antibodies.Mice caused in the 0th, the 14 and 28 day Pam3Cys.WNV001 with not modified WNV001 of independent PBS, 22mg (SEQID NO:771) or 30 μ g exempt from.In the 35th day, by direct ELISA characterization, and measure the serum that derives from indivedual mices to the IgG concentration of synthetic WNV001 peptide.

Figure 73 describes the aminoacid sequence (SEQ ID NOS:691-698) of the EI/EIII junction of west nile virus, Japanese encephalitis virus and dengue virus (serotype 1 to 4).Underline indicate use the STF2 Δ .EIIIs+ that hangs oneself cause the antiserum of exempting from animal identify the west nile virus epitope.This sequence is equivalent to peptide E2-21 (SEQ ID NO:728).

Figure 74 describes the peptide of three palmitylizations.

Figure 75 describes D1 functional domain, D2 functional domain, TLR5 mobilizing function territory and the hypervariable region (D3 functional domain) of flagellin.

Figure 76 describes D1 functional domain, D2 functional domain, TLR5 mobilizing function territory and the hypervariable region (D3 functional domain) (people such as Mi Cang, nature 424,643-650 (2003)) of flagellin.

Figure 77 describes D0, D1, D2 and the D3 functional domain of flagellin, and is arranged in D2 and D3 functional domain and is suitable for the zone (for example, ripe shearing site, HA, M2e) that antigen inserts or replaces.

Figure 78 describes the aminoacid sequence (SEQ ID NO:815) of bacillus pyocyaneus flagellin.Indicate lysine residue with asterisk.

Figure 79 describes the aminoacid sequence (SEQ ID NO:816) of salmonella typhimurium flagellin.Indicate lysine residue with asterisk.

Figure 80 describes Listeria monoeytogenes flagellin (GenBank accession number: aminoacid sequence Q92DW3) (SEQ ID NO:820).Indicate lysine residue with asterisk.

The specific embodiment

Below with more special description feature of the present invention and other details (with step of the present invention, or present with the combination of the present invention's part), and in claim, point out.Should be appreciated that special embodiment of the present invention is to propose in the mode that exemplifies explanation, but not in order to restriction the present invention.Do not departing under the scope of the present invention, characteristics of principle of the present invention can be used for various embodiments.

The present invention be by and large about, make to stimulate individual in immunoreation, the method for compositions of protective immunological reaction for example, and about said composition is and for example by giving the said composition administration individual Therapeutic Method.

" immune response stimulating " is used for this paper and means, and produces at a certain antigenic at least a portion the antibody and the T-cell of the protein portion of hemagglutinin for example described herein (HA) (for example, HA1-1, HA1-2 albumen).Can produce antibody and T-cell, particularly influenza virus albumen at least a portion of influenza virus protein matter (HA of A, B and/or C type influenza virus and M2 albumen).Stimulate individual interior immunoreation to comprise and make it antigen reactive body fluid of (for example virus protein) tool and/or cell immune response.

Can use the method for having set up, assessment the present invention is used for the compositions for the individual interior immunoreactive method of stimulation, stimulates individual interior immunoreactive ability.Be used to measure the present composition and whether comprise in the exemplary method of individual immune response stimulating, by be fit to technology for example the elisa assay measurement be specific to the manufacturing of this antigenic antibody (for example, IgG antibody); Bring out the potential of the antibody-dependency potentiation of supervention infection; Class-macrophage analysis; By using plaque reduction neutralization test (Plaque ReductionNeutralization Test, PRNT ₈₀) neutralization analyzed; And produce ability people such as (, vaccine (Vaccine) 23:4442-4452 (2005)) Pu Nake of serum antibody in the non-human model (for example, mice, rabbit, monkey).

" stimulation protective immunological reaction " is used for this paper and means; the administration present composition; for example hemagglutinin (HA) albumen (for example; HA1-1 as herein described, HA1-2 albumen); cause producing at this proteinic antibody, and make the survival that excites of individual fatal dose from a certain virus protein (for example influenza virus HA) thus.The technology that is used for measuring virus (for example influenza virus) fatal dose for this skill personage known (referring to, for example, WHO/CDS/CSR/NCS2002.5 " WHO is for diagnosis of animal influenza and supervision handbook " World Health Organization (WHO), infectious disease supervision and reaction department, WHO world influenza syllabus (" WHO Manual on Animal Influenza Diagnosis and Surveillance " World HealthOrganization, Dept of Communicable Disease Surveillance and Response, WHOGlobal Influenza Programme); Ha Meng, people such as M.W., J.Clin.Microbiol.26:333-337 (1988); Lee gets, people such as L.J., Am.J.Hyg.27:493-497 (1938); Luo Sai, people such as T., J.Clin.Microbiol.37:937-943 (1999); Wa Ersi, people such as H.H., J.Clin.Microbiol.23:240-245 (1986); Immunologic strategy (Current Protocols in Immunology) now, 19.11.1-19.11.32, Ke carries, people such as R., (the John Wiley ﹠amp of John Willie father and son limited company (2001); Sons, Inc)).The exemplary technology that is used to measure fatal dose can comprise, the various viral dosage of administration are also measured behind administration virus dosage still individual percentage ratio (for example, the LD of survival ₁₀, LD ₂₀, LD ₄₀, LD ₅₀, LD ₆₀, LD ₇₀, LD ₈₀, LD ₉₀).For example, cause the viral fatal dose of 50% death of individual group to be called " LD ₅₀"; Cause the viral fatal dose of 80% death of individual group to be called " LD ₈₀"; Cause the viral fatal dose of 90% death of individual group to be called " LD ₉₀".

For example, (diluent for example is as 8 x 10 can to pass through the various dosage of intranasal administration ³The logarithm of egg-infective dose (EID) and half-log10 dilution liquid), subsequently in about 14 days survival rates of this viral infection, and in individual (for example mice), carry out LD to about 21 days post analysis individualities ₉₀Mensuration.Can be by individual health sign, general behavior (vigor), body weight (body kind loss originally; return back to subsequently and approximate the body weight of this individuality before greatly with viral infection) and through the survival rate of viral infection about 14 to about 21 days post analysis individualities, and analyze protective immunity.

The analysis of the stimulation of protective immunity; can be to analyze in (for example also by utilizing it to the present composition; the proteic part of natural viral is as the protein portion of hemagglutinin) reaction in the antibody that produced, can in and virus protein (for example hemagglutination fibroin) reach with the analysis of the bonded ability of host cell (referring to; for example; immunologic strategy now, 19.11.1-19.11.32, Ke carries; R. wait the people, John Willie father and son limited company (2001)).The analysis of the stimulation of protective immunity also can be passed through, utilize its be measure antibody suppress the analysis of the bonded ability of hemagglutinin and reach (referring to, for example, Bai Neite, people such as F.M., J.exp.Biol.Med.Sci.25:227-233 (1947); Picogram, J.E.J.Immunol.49:87-98 (1944); Immunologic strategy now, 19.11.1-19.11.32, Ke carries, people such as R., John Willie father and son limited company (2001)).

Saltyly believe; the bonded inhibitory action of hemagglutinin is in the antibody (from compositions and form by the inventive method) and the sialic acid binding site (the bonded neutralization of HA) of natural viral hemagglutinin, thus because of stimulating the generation protective immunological reaction to prevent the index of the infected ability of host cell.Bonded inhibition of hemagglutinin or neutralization saltyly believe it is with relevant in order to the immunoreactive ability of the viral fatal dose of protection antagonism.

The bonded neutralization of HA can be passed through, in vitro analyze (referring to, for example, immunologic strategy now, 19.11.1-19.11.32, Ke carries, people such as R., Suppl.42, John Willie father and son limited company (2001) and WHO are for diagnosis of animal influenza and supervision handbook, Robert Webster, people such as R., 28-36,48-54,82-92 page or leaf (2002)) analyze.Exemplary viral neutralization analysis is dependent on the serological specificity combination, and prevents the ability that influenza virus is duplicated in culture (for example, in Martin-Da Bi dog kidney (MDCK) cell line).The letter speech, cell is incubated in 96 orifice plates under the virus of tiring surely in advance exists, and observes the CPE of replication-competent virus in microscopically.For test sera, be prepared into serial dilution serum, and under 37 ℃, cultivated in advance 2 hours with deposit virus, afterwards it is infected mdck cell.Mixture was cultivated 2 hours more in addition, thereafter virus/serum mixture is removed, and displacement is with fresh culture.Make cell growth 4 days.If for a certain given serum dilution, at least about 50% cell death, then the viral growth in hole just is designated as and divides at least about half Kong Zhongyou.Protect an about semicell to avoid the dilution inverse of highest serum of death (in), promptly be considered as neutralization and tire at least about half hole count.

Perhaps, can carry out trace-neutralization and in vitro analyze, to analyze the bonded neutralization of HA.For example, the serum dilution is also cultivated with known virus of tiring as mentioned above in advance, mix with mdck cell again.After cultivating in 2 days, with the cell cleaning and with acetone fixed.Plate is used monoclonal antibody colour generation at influenza nuclear antigen NP according to ELISA.It is to be determined as that microneutralization is tired, about 50% the dilution inverse of highest serum of anti--NP value of reading that its generation is less than the matched group hole gained that only contains virus.

Hemagglutinin inhibition (HAI) is analyzed to be based on and is existed the lip-deep HA antigen of influenza virus can make erythrocyte (RBC) coagulation, and prevents the red blood cell precipitation.With the bonded antibody of sialic acid land specificity of HA, can prevent coagulation and precipitation is taken place.This analysis is to carry out in the 96 hole V base plates that contain new freshly-slaughtered poultry RBC.The virus antigen reserve is tired surely, so that the antigen amount that exists is for doubly excessive with respect to the about 4-that prevents to precipitate required minimum.With test sera (can derive from several species, comprise mice, ferret, poultry or the mankind) be heated to about 56 ℃ so that complement deactivate.Will serum carry out 2-and doubly dilute, and mix with deposit HA through deactivating., cultivated about 30 to about 45 minutes after following 30 minutes in room temperature with the RBC adding and with plate.The result is scored by observing: coagulation causes muddy shape hole, and inhibitory action produces the erythrocyte " button " that is deposited in the bottom, hole.Matched group comprises the RBC group that does not contain HA, and it forms button, and HA and RBC do not contain serum group, and it still keeps muddy shape.The HAI of specific serum sample tire into, prevent the last dilution inverse of coagulation (that is form button).For example, if the 1:128 dilution factor is button, but the 1:256 dilution factor does not present, and then its HAI tires and is about 128.

In an embodiment; the present invention is for making the method for protein that stimulates individual interior protective immunological reaction; it comprises isolates a proteinic part from the natural viral hemagglutinin; and form the step of protein portion thus; wherein this protein portion comprises at least a portion of ball head; and at least a at least a portion that makes ball head in fact still possess the secondary structure of its tertiary structure, and wherein this protein portion lacks film fusion function territory, strides film functional domain and kytoplasm functional domain.The nucleotide sequence of this protein portion of coding is converted in the prokaryotic host cell, and cultivate this prokaryotic host cell and make to stimulate thus individual in the protein of protective immunological reaction.

" a proteinic part " or " protein portion " are used for this paper and mean about the natural viral hemagglutinin, and it is less than the part of any natural viral hemagglutinin of complete natural viral hemagglutinin." natural viral hemagglutinin " is used for this paper and means, as its intact virus hemagglutinin in the form of occurring in nature.

Protein portion can further lack signal sequence.Protein portion can further comprise the sialic acid binding site.

The protein portion that is used for the natural viral hemagglutinin of the present composition and method, can be Orthomyxoviridae family (influenza virus A, B, C), paramyxovirus (parainfluenza virus, respiratory tract amalgamation virus, new castle disease virus, upright all kinds of diseases and ailments poison (Nipah), Measles virus, canine distemper virus, Sendai virus), Reoviridae (rotavirus), picodnavirus section (human small DNA virus, the pig small DNA virus), orthopoxvirus (monkey pox virus, mouse pox virus), flaviviridae (west nile virus, Japanese encephalitis, the Saint Louis, the tired paddy of China ink, Kun Jin (Kunjin) virus), fowlpox virus (chicken fowlpox virus), upright all kinds of diseases and ailments poison (people such as paddy labor nurse V., J.Virol., 80:7546-54 (2006)); Canine distemper virus (people such as Xin Getan K., J Gen Virol., 87:1635-42 (2006)); New castle disease virus (people such as enlightening Li Ou O.S., JGen Virol., 86:1759-69 (2005); Reach people such as Mei Lasen V.R., J Virol., 78:13053-61 (2004); People such as Deng R., virusology (Virology), 204:17-26; (1994)), measles people such as (, J Virol., 78:9051-63 (2004)) the husky N. of horse, Sendai virus (people such as Thomas west M., FEBS Lett., 11:56-60 (2003)), human parainfluenza (people such as bohr holder M., J Virol., 79:2383-92 (2005); People such as crane road M., virusology, 213:190-203 (1995); People such as ripple plug T., virusology, 209:654-7 (1995); And people such as this T. of waterfall, J Virol., 66:7597-600 (1992)) a part.

The viral hemagglutination element (for example, influenza virus A, influenza virus B and influenza virus C viral hemagglutination element) part (" protein portion "), can comprise at least a member who is called the cohort that protein portion that " HA1-1 ", " HA1-2 " reach " HA1-3 " forms by this paper that is selected from.

" HA1-1 " is used for the protein portion that this paper means the viral hemagglutination element, it comprises at least about a β-interlayer that comprises substrate binding site (β-sandwich), it comprises at least about two β-flaps and (β-sheet), is positioned at the extra little β-interlayer of this molecule bottom at least about two to about three short alpha-helixs, at least one little β-flap and at least one, and at least four disulfide bond.β-the interlayer that comprises the substrate binding site of HA1-1 comprises at least about four β-thighs as top end flap, and at least about three to about four β-thighs as bottom flap.HA1-1 part be to be positioned at the next door that it comprises the β-interlayer of substrate binding site at least about an alpha-helix, and be to be positioned at the bottom that it comprises the β-interlayer of substrate binding site at least about about one to about two.Little β-interlayer of HA1-1 can comprise in each β-flap at least about two to about three β-thighs; Or comprise about three to about four β-thighs.Exemplary HA1-1 protein portion comprises SEQ ID NOS:8,11,14,17,20,38,40,45,47,49,179,180,181 and 182.

" HA1-2 " is used for the protein portion that this paper means the viral hemagglutination element, it comprise at least about one it comprise the β-interlayer of substrate binding site, at least about two to about three short alpha-helixs, be positioned at the little β-flap of this molecule bottom and at least about two disulfide bond at least about one.β-thigh in the viral hemagglutination element can comprise about 2 to about 15 aminoacid.Little β-folding thigh can comprise about two aminoacid; Or about two to about three aminoacid; Or about two to about four aminoacid; Or about two to about five amino acid.Little β-flap can comprise about two to about three β-thighs; Or about two to about four β-thighs.β-the interlayer that comprises the HA1-2 substrate binding site can further comprise at least about four β-thighs as top end flap, and at least about three to about four β-thighs as bottom flap.HA1-2 part be to be positioned at the next door that it comprises the β-interlayer of substrate binding site at least about an alpha-helix, and be to be positioned at the bottom that it comprises the β-interlayer of substrate binding site at least about one to about two.Exemplary HA1-2 protein portion comprises SEQ ID NOS:9,12,15,18,21,24,26,28,30,32,39,41,46,48 and 50.

" HA1-3 " is used for the protein portion that this paper means the viral hemagglutination element, it comprise at least about one it comprise the β-interlayer of substrate binding site, at least about two short alpha-helixs and at least about a disulfide bond." β-interlayer " is used for this paper and means the β-flap group that forms at least about an interplay layer at least about two groups." substrate binding site " is used for this paper is to mean any part that has with a certain molecular action or bonded natural viral hemagglutinin about β-interlayer.For example, comprise the β-interlayer of the substrate binding site of this part, can comprise and the bonded part of sialic acid.β-the interlayer that comprises the HA1-3 substrate binding site can further comprise at least about four β-thighs as top end flap, and at least about three β-thighs as bottom flap.At least one alpha-helix of HA1-1 part be the next door that is positioned at the β-interlayer that comprises substrate binding site, and at least one other alpha-helix is the bottom that is positioned at the β-interlayer that comprises substrate binding site.Short alpha-helix can comprise in an alpha-helix and is less than about 5 corners (2,3,4,5 corners).Alpha-helix in the viral hemagglutination element can have one to about 15 times corners; Or about two to about 15 corners.Exemplary HA1-3 protein portion comprises SEQ ID NOS:10,13,16,19,22,25,27,29,31 and 33.

" sialic acid binding site " be used for this paper about derive from the proteinic part of natural viral hemagglutinin, mean have can with the part of the protein portion of the ability of sialic acid residues effect.

" sialic acid binding site " is used for this paper and also refers to " sialic acid is in conjunction with the territory ".

" at least a portion " is used for this paper and means, a certain composition (for example, ball head, secondary structure) or any part of molecule (for example, the outer functional domain (M2e) of protein, antigen, clock sample receptor, peptide, flagellin, HA, substrate 2 albumen (M2), substrate 2 born of the same parents); Or whole should the composition or this molecule." at least a portion " is used for this paper and also is called " fragment ".

" at least a portion " about flagellin (for example is used for this paper, fljB/STF2, escherichia coli fliC, Munich Salmonella fliC) be meant, can cause any part (for example, motif (motif) C in the flagellin for the intracellular signal transmission of clock sample (Toll) receptor; Motif N; Functional domain 1,2,3) or flagellin integral body.

Single polypeptide can present several type secondary structures.Under the mutual effect of no any stabilization glue, polypeptide can be taked the random coil configuration.Yet, secondary structure (for example Ah cutting down (α)-spiral and β (β)-thigh) but a stable protein or a proteinic part.Side direction between β-thigh is associated and is formed β-flap (also being called " beta sheet " in this paper).Secondary structure can be positioned at the surface of this part, protein or native protein (for example, viral hemagglutination element, flagellin, M2e).Proteinic tertiary structure is that three-Wei of amino acid residue arranges.With respect to secondary structure, it is to stablize by (for example) hydrogen bond, alpha-helix, β-thigh, and tertiary structure causes because of the hydrophobic interaction between the non-polar sidechain of this part, protein or natural viral hemagglutinin.Hydrophobic interaction makes the spiral thigh in the random coil be tight internal stent.Proteinic size and shape can be according to one grade amino acid sequence, and the number of secondary structure, size and arrangement.

" ball head " is used for this paper and means the proteinic part that it comprises the natural viral hemagglutinin of receptor or sialic acid land." ball head " also is called " spherical functional domain " in this paper.Based on as people such as (for example) Wilson I.A., Nature 289:366-373 (1981); Old, people such as J., cell (Cell) 95:409-417 (1998); Breathe out people such as Y., The EMBO Journal 21:865-875 (2002); The Ruse that, R.J. virusology 325:287-296 (2004); And Ke Kesi, N.J. wait the people, in: microbiology and the infected by microbes (Toply and Wilson ' s Microbiology and MicrobialInfections) with Wilson founds in Top, people such as BWJ Ma Di are compiling, volume 1 (the 9th edition) New York, NY, the Oxford University publishes (OxfordUniv.Press), the 32nd chapter, p.634 (1998) described x-radiocrystallography determines the ball head of viral hemagglutination fibroin matter.The ball head of natural viral hemagglutinin is the composition of the HA1 subunit of (for example) influenza virus haemagglutinin.Except the receptors bind functional domain, ball head can comprise as people such as (for example) Kazakhstan Y., The EMBO Journal 21:865-875 (2002) described E-subfunction territory and F ^-The subfunction territory.

Words and phrases " make ball head in fact still possess its tertiary structure " to be used for this paper mean the ball head of keeping the natural viral hemagglutinin be enough to stimulate individual in the tertiary structure of protective immunological reaction.

The film fusion function territory of viral hemagglutination element is in conjunction with the zone (relating to the combination of viral hemagglutination element) of host cell in the viral hemagglutination element.The film functional domain of striding of viral hemagglutination element is to cross over the part of viromembrane in the viral hemagglutination element.The kytoplasm functional domain of viral hemagglutination element is to be positioned at the lip-deep part of virocyte matter in the viral hemagglutination element.

The proteinic part of natural viral hemagglutinin (this paper also is called " protein portion ") can further lack signal sequence.The part that is used for the ball head of methods described herein can comprise at least a portion of at least one secondary structure, this secondary structure comprise at least a portion of at least one beta sheet-; At least one α spiral and/or at least a member who is selected from the cohort of forming by salt bridge, leucine zipper and zinc fingers.The ball head part can further comprise at least about a disulfide bond, at least about two disulfide bond, at least about three disulfide bond, at least about four disulfide bond, at least about five disulfide bond and at least about six disulfide bond.

Make to stimulate individual in the method for protein of protective immunological reaction can comprise further that at least one is selected from the nucleotide sequence of the amino acid residue of the cohort of being made up of hydrophilic amino-acid residue, polar amino acid residue and neutral amino acid residue to encode, replace the step of the nucleotide sequence of at least one hydrophobic amino acid residues in this protein portion of coding.Substituted hydrophobic amino acid residues can comprise at least a member who is selected from the cohort of being made up of phenylalanine residue, trp residue and tyrosine residue.Replace the polar amino acid residue of hydrophobic amino acid residues, can comprise at least a member who is selected from the cohort of forming by asparagicacid residue and glutaminic acid residue.

The proteinic part of natural viral hemagglutinin can be the part of natural viral hemagglutinin protein (for example influenza A, B and C).Influenza A virus hemagglutinin protein can be at least a member who is selected from the cohort of being made up of H1, H2, H3, H5, H7 and H9.

The host cell that is used for methods described herein can be a kind of prokaryotic host cell.Prokaryotic host cell can be at least a member who is selected from the cohort of being made up of escherichia coli prokaryotic host cell, Rhodopseudomonas prokaryotic host cell, bacillus prokaryotic host cell, salmonella spp prokaryotic host cell and pseudomonas fluorescens prokaryotic host cell.

Make the method for protein that stimulates individual interior protective immunological reaction and can further comprise the step that prokaryotic host cell is transformed with molecular chaperones (chaperone) nucleotide sequence.The molecular chaperones nucleotide sequence can be at least a member who is selected from the cohort of being made up of groES-groEL molecular chaperones, dnaK-dnaJ-grpE molecular chaperones, groES-groEL-tig molecular chaperones and tig molecular chaperones.

Make the method for protein that stimulates individual interior protective immunological reaction and can comprise further that at least a portion with clock sample receptor (TLR) agonist merges to this proteinic step.The nucleotide sequence of at least a portion of coding clock sample receptor stimulating agent can be linked to the nucleotide sequence of the protein portion of coding viral hemagglutination element through operability.Connexon (for example peptide connexon) can be between the part of clock sample receptor stimulating agent and viral hemagglutination element.

Clock sample receptor is meant a kind of and receptor protein family Drosophila melanogaster (Drosophila melangogaster) Bristol albumen homology.Clock sample receptor is with rich leucine repeat function territory outside the born of the same parents, and strides film citation receptor protein with the I type that the interior functional domain of the born of the same parents of white plain 1 receptor homolog that is situated between is a feature.Clock sample receptor comprises TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11 and TLR12.

" agonist " is used for this paper is the molecule that means activation TLR citation approach about TLR.TLR citation approach is a kind of by signaling path in the cell that utilized by TLR part or the activatory specific T LR of TLR agonist.Approach is to be utilized by TLR in the general born of the same parents, and comprises (for example) NF-κ B, Jun N-end kinases and mitogen-activatory protein kinase.Clock sample receptor stimulating agent can comprise at least a agonist by TLR1 that is selected from, TLR2 agonist (Pam3Cys for example, Pam2Cys, bacterial lipoprotein), TLR3 agonist (for example dsRNA), TLR4 agonist (for example bacteria lipopolysaccharide), TLR5 agonist (for example dynein), the TLR6 agonist, the TLR7 agonist, the TLR8 agonist, TLR9 agonist (for example without the fat DNA motif that methylates), the TLR10 agonist, the member of the cohort that TLR 11 agonist and TLR 12 agonist are formed.

It is that the clock sample receptor stimulating agent of at least a portion of clock sample receptor stimulating agent is formed that the clock sample receptor stimulating agent that is used for the inventive method and compositions can be a kind of, wherein this clock sample receptor stimulating agent is formed and is comprised at least one cysteine residues, it is arranged in natural clock sample receptor stimulating agent and locating of cysteine residues do not occur, makes this clock sample receptor stimulating agent form activation clock sample receptor thus.

The TLR4 part (for example TLR4 agonist) that is used for the present composition and method can comprise at least a being selected from by SEQ ID NOS:359-406 (referring to, PCT/US 2006/002906/WO2006/083706; PCT/US 2006/003285/WO 2006/083792; PCT/US 2006/041865; PCT/US 2006/042051) member of the cohort formed.

GGKSGRTG (SEQ ID NO：359)

KGYDWLVVG (SEQ ID NO：360)

EDMVYRIGVP (SEQ ID NO：361)

VKLSGS (SEQ ID NO：362)

GMLSLALF (SEQ ID NO：363)

CVVGSVR (SEQ ID NO：364)

IVRGCLGW (SEQ ID NO：365)

AAEERTLG (SEQ ID NO：366)

WARVVGWLR (SEQ ID NO：367)

SEGYRLFGG (SEQ ID NO：368)

LVGGVVRRGS (SEQ ID NO：369)

GRVNDLWLAA (SEQ ID NO：370)

SGWMLWREGS (SEQ ID NO：371)

ERMEDRGGDL (SEQ ID NO：372)

KLCCFTECM (SEQ ID NO：373)

AVGSMERGRG (SEQ ID NO：374)

RDWVGGDLV (SEQ ID NO：375)

FFEVAKISQQ (SEQ ID NO：376)

WWYWC (SEQ ID NO：377)

MHLCSHA (SEQ ID NO：378)

WLFRRIG (SEQ ID NO：379)

YWFWRIG (SEQ ID NO：380)

MHLYCIA (SEQ ID NO：381)

WPLFPWIV (SEQ ID NO：382)

DMRSHAR (SEQ ID NO：383)

MHLCTHA (SEQ ID NO：384)

NLFPFY (SEQ ID NO：385)

MHLCTRA (SEQ ID NO：386)

RHLWYHA (SEQ ID NO：387)

WPFSAYW (SEQ ID NO：388)

WYLRGS (SEQ ID NO：389)

GKGTDLG (SEQ ID NO：390)

IFVRMR (SEQ ID NO：391)

WLFRPVF (SEQ ID NO：392)

FLGWLMG (SEQ ID NO：393)

MHLWHHA (SEQ ID NO：394)

WWFPWKA (SEQ ID NO：395)

WYLPWLG (SEQ ID NO：396)

WPFPRTF (SEQ ID NO：397)

WPFPAYW (SEQ ID NO：398)

FLGLRWL (SEQ ID NO：399)

SRTDVGVLEV (SEQ ID NO：400)

REKVSRGDKG (SEQ ID NO：401)

DWDAVESEYM (SEQ ID NO：402)

VSSAQEVRVP (SEQ ID NO：403)

LTYGGLEALG (SEQ ID NO：404)

VEEYSSSGVS (SEQ ID NO：405)

VCEVSDSVMA (SEQ ID NO：406)

The TLR2 part (for example TLR2 agonist) that is used for the present composition and method also can comprise at least a being selected from by SEQ ID NOS:455-494 (referring to, PCT/US 2006/002906/WO2006/083706; PCT/US 2006/003285/WO 2006/083792; PCT/US 2006/041865; PCT/US 2006/042051) member of the cohort formed.

NPPTT (SEQ ID NO：455)

MRRIL (SEQ ID NO：456)

MISS (SEQ ID NO：457)

RGGSK (SEQ ID NO：458)

RGGF (SEQ ID NO：459)

NRTVF (SEQ ID NO：460)

NRFGL (SEQ ID NO：461)

SRHGR (SEQ ID NO：462)

IMRHP (SEQ ID NO：463)

EVCAP (SEQ ID NO：464)

ACGVY (SEQ ID NO：465)

CGPKL (SEQ ID NO：466)

AGCFS (SEQ ID NO：467)

SGGLF (SEQ ID NO：468)

AVRLS (SEQ ID NO：469)

GGKLS (SEQ ID NO：470)

VSEGV (SEQ ID NO：471)

KCQSF (SEQ ID NO：472)

FCGLG (SEQ ID NO：473)

PESGV (SEQ ID NO：474)

DPDSG (SEQ ID NO：475)

IGRFR (SEQ ID NO：476)

MGTLP (SEQ ID NO：477)

ADTHQ (SEQ ID NO：478)

HLLPG (SEQ ID NO：479)

GPLLH (SEQ ID NO：480)

NYRRW (SEQ ID NO：481)

LRQGR (SEQ ID NO：482)

IMWFP (SEQ ID NO：483)

RVVAP (SEQ ID NO：484)

IHVVP (SEQ ID NO：485)

MFGVP (SEQ ID NO：486)

CVWLQ (SEQ ID NO：487)

IYKLA (SEQ ID NO：488)

KGWF (SEQ ID NO：489)

KYMPH (SEQ ID NO：490)

VGKND (SEQ ID NO：491)

THKPK (SEQ ID NO：492)

SHIAL (SEQ ID NO：493)

AWAGT (SEQ ID NO：494)

TLR2 part (for example TLR2 agonist) also can comprise at least a flagellin modified protein FlmB that is selected from by crescent handle bacillus (Caulobacter crescentus); Antibacterial III type excretory system albumen; The intrusion albumen of Salmonella; The 4th type pili source of students albumen (fimbrialbiogenesis protein) of pseudomonas (PilX); Salmonella SciJ albumen; Streptomycete infer conformability memebrane protein (intergral membrane protein); The memebrane protein of pseudomonas; The adhesin of Hemophilus pertussis (adhesin); The PEPB B of vibrio cholera; The virulence induction albumen of Bo Deshi bacillus; Meningococcus infer the conformability memebrane protein; Flagellum biosynthesis albumen FliR of fusobacterium and the fusant of FlhB; The outer membrane protein of acinetobacter (porin); The flagellum biosynthesis albumen FlhF of Helicobacterium; The ompA related protein of xanthomonas (Xanthomonas); The omp2a porin of Brucella; Porin/the pili of inferring of Salmonella is assembled albumen (LHrE); The wbdk of Salmonella; Relate to the glycosyl transferase that the LPS biology is examined; Salmonella is inferred at least a portion of the member of the cohort that permease forms.

TLR2 part (for example TLR agonist) can comprise at least a being selected from by lipoprotein/lipopeptid class (various pathogen); Peptidoglycan (gram-positive bacteria); Lipoteichoic acid (lipoteichoic acid) (gram-positive bacteria); Fat AM (lipoarabinomannan) (Mycobacterium); Phenol-solubility modulin (modulin) (staphylococcus epidermidis); Glycosyl lipositol class (schizotrypanum cruzi); Glycolipid class (treponema maltophilum (Treponema maltophilum)); Porin (Neisseria); At least a portion of the member of the cohort that zymosan (fungus) and atypia LPS (leptospira interrogans (Leptospira interrogans) and porphyromonas gingivalis (Porphyromonas gingivalis)) are formed.

TLR2 part (for example TLR2 agonist) also can comprise, at least a being selected from by SEQ ID NOS:495-497 (referring to, PCT/US 2006/002906/WO 2006/083706; PCT/US2006/003285/WO 2006/083792; PCT/US 2006/041865; PCT/US 2006/042051) at least a portion of the member of the cohort formed.

KGGVGPVRRSSRLRRTTQPG (SEQ ID NO：495)

GRRGLCRGCRTRGRIKQLQSAHK (SEQ ID NO：496)

RWGYHLRDRKYKGVRSHKGVPR (SEQ ID NO：497)

The TLR2 agonist can comprise at least a portion of bacterial lipoprotein (BLP).

The TLR2 agonist can be a kind of bacterial lipoprotein; Pam2Cys (S-[2 for example; two (the palmityl oxygen base) propyl group of 3-] cysteine), Pam3Cys ([palmityl]-Cys ((RS)-2,3-two (palmityl oxygen base)-propylcysteine) or bacillus pyocyaneus OprI lipoprotein (OprI).Exemplary OprI lipoprotein comprises SEQ IDNO:782, and it is encoded by SEQ ID NO:783.A kind of exemplary escherichia coli lipoprotein of the present invention that is used for as herein described is the SEQ ID NO:784 by SEQ ID NO:785 coding.The bacterial lipoprotein of activation TLR citation approach (TLR2 agonist) is the bacterioprotein (Ao Mu carries, people such as K.O., J.Biol.Chem.280:36616-36625 (2005)) that comprises palmitoleic acid.For example, the expression of SEQ ID NOS:783 and 785 in bacterial expression system (for example escherichia coli), cause with palmitoleic acid partly add to become protein (for example, SEQ ID NOS:782 and 784) on the cysteine residues, produces the TLR2 agonist that is used for the present composition, fusion rotein and polypeptide thus.Via the sulfydryl that the DG group is added to cysteine (for example cysteine 21 of SEQ ID NO:784); subsequently signal sequence is sheared; and the 3rd acyl chain is added to the free state N-end group group of same cysteine; can in antibacterial, make three palmityl lipoproteins (also being called three acyl groups-lipoprotein) (Sang Kalan; K. wait the people; J.Biol.Chem.269:19706 (1994)), produce three palmityl peptides (TLR2 agonist) shown in (for example) Figure 74.

Clock sample receptor stimulating agent in the present composition can further comprise the cysteine residues of the end amino acid of N-terminal end amino acid that at least one is positioned at clock sample receptor stimulating agent and/or c-terminus.For example, SEQ ID NO:359 can comprise further that at least one exists and the bonded cysteine residues of aminoterminal glycine residue peptide bond, and/or at least one exists and the bonded cysteine residues of c-terminus glycine residue peptide bond; SEQ ID NO:360 can comprise further that at least one exists and the bonded cysteine residues of aminoterminal lysine residue peptide bond, and/or at least one exists and the bonded cysteine residues of c-terminus glycine residue peptide bond; SEQ ID NO:361 can comprise further that at least one exists and the bonded cysteine residues of aminoterminal glutaminic acid residue peptide bond, and/or at least one exists and the bonded cysteine residues of c-terminus proline residue peptide bond.

The TLR5 agonist that is used for the inventive method can comprise at least a portion of flagellin.Flagellin for can activate TLR5 pathogen-connection molecule pattern (pathogen-associated molecular pattern, PAMP).

Flagellin in compositions described herein and the method can be at least a portion of salmonella typhimurium flagellin (Genbank accession number AF045151); Be selected from least a portion of the salmonella typhimurium flagellin of the cohort of being formed by SEQ ID NO:498, SEQ IDNO:812, SEQ ID NO:816 and SEQ ID NO:500; At least a portion of Munich Salmonella flagellin (Genbank accession number AB028476), it comprises at least a portion of SEQ ID NO:504 and SEQ ID NO:813; At least a portion of bacillus pyocyaneus flagellin, it comprises at least a portion of SEQ ID NO:815; At least a portion of Listeria monoeytogenes flagellin, it comprises at least a portion of SEQ ID NO:820; The proteic at least a portion of coli flagellum, it comprises at least a portion of SEQ ID NO:502 and SEQ ID NO:814; At least a portion of yersinia flagellin; And at least a portion of Campylobacter flagellin.

The flagellin that is used for the present composition also can comprise the polypeptide of SEQ ID NO:498, SEQ ID NO:500, SEQ ID NO:504 and SEQ ID NO:502; At least a portion of at least a portion of at least a portion of SEQ ID NO:498, SEQ ID NO:500, SEQ ID NO:504 and at least a portion of SEQ IDNO:502; By SEQ ID NO:499, SEQ ID NO:501, SEQ ID NO:505 and SEQ ID NO:503 encoded polypeptide; Reach at least a portion by SEQ ID NO:499, SEQ ID NO:501, SEQ ID NO:505 and SEQ ID NO:503 encoded polypeptide.

The flagellin that is used for the present composition and method can lack at least a portion of hinge region.Hinge region is the hypervariable region of flagellin.The hinge region of flagellin in this paper also be called " D3 functional domain or zone ", " propeller functional domain or zone ", " hypervariable region or zone " reaches " domain of variation or zone ".The hinge region of " shortage " flagellin means at least one aminoacid of the hinge region that is contained in flagellin, or at least one this at least one amino acid whose nucleic acid codon of encoding, and is not present in the flagellin.The example of hinge region comprises the amino acid/11 76-415 of SEQ ID NO:498, and it is coded by the nucleic acid 528-1245 of SEQ ID NO:499; The amino acid/11 74-422 of SEQ ID NO:502, it is coded by the nucleic acid 522-1266 of SEQ ID NO:503; Or the amino acid/11 73-464 of SEQ ID NO:504, it is coded by the nucleic acid 519-1392 of the nucleic acid of SEQ ID NO:505.Therefore, as if the flagellin disappearance of amino acid/11 76-415 from SEQ ID NO:498, then this flagellin will lack hinge region.The flagellin that lacks at least a portion of hinge region also is called in this paper " the truncate version " of flagellin.

" at least a portion of hinge region " is used for any part that this paper means the hinge region of flagellin, or whole hinge region." at least a portion of hinge region " also refers to " hinge region fragment " in this paper.At least a portion of the hinge region of fljB/STF2 can be the aminoacid 200-300 of (for example) SEQ ID NO:498.Therefore, if aminoacid 200-300 from SEQ ID NO:498 disappearance, then becomes the aminoacid sequence of STF2 will lack the part of hinge region.

Perhaps, can be with at least a portion of natural flagellin, so that few part displacement of artificial hinge region." natural " means this aminoacid sequence about the flagellin aminoacid sequence and is present in natural flagellin, (for example, salmonella typhimurium (S.typhimurium) flagellin, Munich Salmonella (S.muenchin) flagellin, escherichia coli (E.coli) flagellin).Naturally occurring hinge region is the hinge region that is present in the natural flagellin.For example, the amino acid/11 73-464 of the amino acid/11 76-415 of SEQ ID NO:498, the amino acid/11 74-422 of SEQID NO:502 and SEQ ID NO:504 is equivalent to the natural hinge of STF2, escherichia coli fliC and Munich Salmonella flagellin (fliC) respectively." artificial " is used for this paper and means about the hinge region of flagellin, inserts any hinge region that contains or once contained natural hinge in the natural flagellin.

The hinge region disappearance of flagellin can be reached with antigen (for example HA1-1, HA1-2, ripe shearing site) displacement, and the construct that is become is merged to another kind of antigen (for example 4xM2e).

Artificial hinge region can be used for lacking the natural flagellin of at least a portion of hinge region, its can help the carboxyl of flagellin-with amino terminal in order to the bonded interaction of TLR5, and therefore activate the inherent signal pipeline of TLR5.Lacking the flagellin of at least a portion of hinge region, is to name to add " Δ " after this flagellin title.For example, lacking the STF2 (for example SEQ ID NO:498) of at least a portion of hinge region, is to be called " STF2 Δ " or " fljB/STF2 Δ " (for example SEQ ID NO:500).

The flagellin that is used for the present composition and method can be at least a portion of flagellin, and wherein this flagellin composition comprises at least one cysteine residues, and makes this flagellin composition activation clock sample receptor 5 thus; It is the flagellin composition of at least a portion of flagellin, and wherein at least one lysine of this flagellin composition is replaced by at least one arginine, makes flagellin composition activation clock sample receptor 5 thus; It is the flagellin composition of at least a portion of flagellin, and wherein at least one lysine of this flagellin composition is replaced by at least one serine, makes flagellin composition activation clock sample receptor 5 thus; It is the flagellin composition of at least a portion of flagellin, and wherein at least one lysine of this flagellin composition is replaced by at least one histidine, makes flagellin composition activation clock sample receptor 5 thus, and is as described herein.

Can be by TLR agonist operability be linked to this protein portion, and produce recombination fusion protein (for example, HA1-1, HA1-2)." fusion rotein " is used for this paper and means the protein that produces from least two kinds of similar or different compositions (for example, the protein portion of HA and TLR agonist).Fusion rotein can produce or produce with chemical bond by recombination form.In an embodiment, the fusion rotein of the protein part of HA and TLR agonist (for example, SEQ ID NOS:89-92; 95; 151-160,177,209; 210 and 211) can with the moving agent of TLR and the proteic fusion rotein of M2e (for example; SEQ ID NOS:528,587,589 and 591) mix or together throw and give; be administered to individuality with immune response stimulating, for example protective immunological reaction.

Fusion rotein of the present invention can be disconnected and named by forming with ". " of this fusion rotein.For example, " STF2.HA1-2 " is meant and comprises a STF2 protein and the proteinic fusion rotein of HA1-2; And " STF2 Δ .HA1-2 " is meant and comprises a STF2 protein and the proteinic fusion rotein of HA1-2 that does not contain hinge region.Exemplary fusion rotein of the present invention comprises SEQ ID NOS:89-92,95,151-160,177,209,210 and 211.

Fusion rotein can comprise (for example) two, three, four, five, six or multiple clock sample receptor stimulating agent (for example, flagellin), with two, three, four, five, six or multiple antigen (for example, protein portion such as HA1-1, HA1-2).When two or multiple TLR agonist and two or multiple proteins when partly comprising fusion rotein of the present invention, they also are called " polymer ".For example, the polymer of HA1-1 can be four HA1-1 sequences, and it is known as 4xHA1-1 in this paper.Equally, " 2xHA1-1 " is the polymer be made up of two HA1-1 sequences (referring to, SEQ ID NOS:342-346 and 348 for example).

Fusion rotein of the present invention can further comprise, connexon between between at least one other composition (for example HA1-1, HA1-2) of at least one composition (for example TLR agonist) of fusion rotein and fusion rotein, connexon between between extremely a small amount of individual similar composition (for example HA1-1, HA1-2) of fusion rotein, or its combination in any." connexon " is used for this paper and means about fusion rotein of the present invention, so that each of fusion rotein formed not direct banded mode, and the junctional complex between between each composition of fusion rotein.For example, the different piece (for example, protein portion such as HA1-1, HA1-2, antigen) that the part of fusion rotein (for example flagellin) can be through being connected to fusion rotein.At least two or a plurality of similar or similar composition (for example, two flagellins can comprise further that between the connexon between each flagellin, perhaps two HA protein can further comprise the connexon between between each HA protein) that can connect equally, fusion rotein.

(or in addition alternatively) in addition, fusion rotein of the present invention can comprise between each of fusion rotein formed between similar or similar composition to fusion rotein the connexon combination.For example, fusion rotein can comprise at least two kinds of TLR agonist, and it further comprises the connexon between between (for example) two or multiple flagellin; The protein portion of at least two HA, it further comprises the connexon between between they; Connexon between between the another kind of different compositions (for example, the protein portion of HA) of a kind of composition (for example, flagellin) of fusion rotein and fusion rotein, or its combination in any.

Connexon can be the aminoacid connexon.The aminoacid connexon can comprise synthetic or natural amino acid residue.The aminoacid connexon that is used for fusion rotein of the present invention can comprise at least a member who is selected from by lysine residue, paddy lysine residue, the silk cohort that lysine residue and smart lysine residue became.The aminoacid connexon can comprise (for example) SEQ ID NOS:521,523,524 and 526, respectively by SEQ ID NOS:520,522,525 and 527 nucleic acid sequence encoding.

The clock sample receptor stimulating agent of fusion rotein of the present invention can merge extremely, c-terminus, aminoterminal or the carboxyl of the protein portion of HA (for example HA1-1, HA1-2 or other antigen are as the outer functional domain of substrate 2 proteic born of the same parents, ripe shearing site) and aminoterminal the two.

Fusion rotein can be by protein portion (or other antigen with HA, the ripe shearing site of HA for example) merges in four zones ( zone 1,2,3 and 4) (it identifies based on the crystal structure (PDB:1UCU) of flagellin, referring to Figure 77) to flagellin one of them and produce.

Zone 1 is TIAL (SEQ ID NO:823) ...-... GLG (95-111 of SEQ ID NO:841).Corresponding residue for salmonella typhimurium fljB construct is TTLD (SEQ ID NO:824) ...-... GTN (196-216 of SEQ ID NO:841).The two β thighs (unfolding peptides of the ditch of GTDQKID (SEQ ID NO:825) and NGEVTL (SEQ ID NO:826) of this zone for being arranged in (SEQ ID NO:841).This peptide can be simulated the configuration of the ripe shearing site peptide of wild type among the HA0 with the replacement of a certain antigen (for example ripe shearing site of HA).Can comprise by substituted exemplary aminoacid: flagellin residue SGLDDAAIKAAT (SEQ ID NO:827) (201-212 of SEQ ID NO:841), through ripe shearing site residue: RGIFGAIAGFIE (SEQ ID NO:828), it is equivalent to the A/H3N2 hypotype, or RGLFGAIAGFIE (SEQ ID NO:803), it is equivalent to derive from the ripe shearing site residue of A/H2N1, A/H1N1, A/H5N1 hypotype, or through RGFFGAIAGFLE (SEQ ID NO:805), it is equivalent to the ripe shearing site residue of influenza B HA and replaces.

The zone 2 of Salmonella flagellin is the little ring GTG (238-240 of SEQ ID NO:841) (referring to Figure 77) in the 1UCU structure.Corresponding ring among the Salmonella fljB is GADAA (SEQID NO:829) (244-248 of SEQ ID NO:841).In this ring, insert antigen (for example protein portion of HA, ripe shearing site peptide), or replace whole ring with antigen (for example protein portion of HA, ripe shearing site peptide), should keep the stretching, extension ring structure of ripe shearing site peptide and natural HA molecular association.

Zone 3 for be present on 1 the opposition side of zone than macro ring (referring to Figure 77).This ring can be substituted with zone 1 simultaneously, and produces two copy antigens (for example protein portion of HA, ripe shearing site peptide).This ring originates in AGGA (SEQ ID NO:830), and ends in PATA (SEQ ID NO:831) (259-274 of SEQ ID NO:841).Corresponding Salmonella fljB sequence is AAGA (SEQ IDNO:832...-...ATTK (SEQ ID NO:833) (266-281 of SEQ ID NO:841).Can be with sequence A GATKTTMPAGA (SEQ ID NO:834) (267-278 of SEQ ID NO:841) displacement with antigen (for example protein portion of HA, ripe shearing site peptide).

Zone 4 is the ring (VTGTG (SEQ ID NO:835)) (referring to Figure 77) of the short alpha-helix (TEAKAALTAA (SEQ ID NO:836)) in the binding 1UCU structure with β-thigh (ASVVKMSYTDN (SEQ ID NO:837)).Corresponding ring among the Salmonella fljB is long ring VDATDANGA (SEQID NO:838 (307-315 of SEQ ID NO:841).Antigen (for example protein portion of HA, ripe shearing site peptide) can be inserted or replace this zone.

Can insert or use the multicopy permutating column of antigen (for example, ripe shearing site peptide) to be shown in peptide in aforementioned four zones.Preferably, displacement should be positioned at zone 1 and zone 3.

The exemplary sequence of the ripe shearing site peptide of HA, series is shown in down:

The sequence hypotype

NVPEKQTRGIFGAIAGFIE A/H3N2(SEQ ID NO：800)

NVPQIESRGLFGAIAGFIE A/H2N1(SEQ ID NO：801)

NIPSIQSRGLFGAIAGFIE A/H1N1(SEQ ID NO：802)

RERRRKKRGLFGAIAGFIE A/H5N1(SEQ ID NO：839)

PAKLLKERGFFGAIAGFLE B/HA(SEQ ID NO：804)

X-ray pattern (1UCU) is calibrated with the sequence of SEQ ID NO:841

It is overlapping that 75.1% homogeneity is present in 506 residues; Scoring: 1703.0;

Space frequency: 2.6%

1UCU 1 AQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANRFTANIKGL

sfla 2 AQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANRFTANIKGL

************************************************************

1UCU 61 TQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLN

sfla 62 TQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLN

************************************************************

1UCU 121 EIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDTLNVQQKYKV

sfla 122 EIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDSLNVQKAYDV

************************************************** **** * *\

UCU 181 SDTAATVTGYAD--TTI---ALDNSTFKASATGLGGTDQKIDGDLKFDDTTGKYYAKVTV

sfla 182 KDTAVTTKAYANNGTTLDVSGLDDAAIKAATGGTNGTASVTGGAVKFDADNNKYFVTIGG

*** * ** ** ** ** * ** * *** **

1UCU 236 TGGT--GKDGYYEVSVDKTNGEVTLAGGATSPLTGGLPATATEDVKNVQVANADLTEAKA

sfla 242 FTGADAAKNGDYEVNV-ATDGTVTLAAGATKTTMPAGATTKTEVQELKDTPAVVSADAKN

* * * *** * * * **** *** * ** **

1UCU 294 ALTAAGVTGT----ASVVKMSYTDNNGKTIDGGLAVKVGDDYYSATQNK-DGSISINTTK

sfla 301 ALIAGGVDATDANGAELVKMSYTDKNGKTIEGGYALKAGDKYYAADYDEATGAIKAKTTS

** * ** * ******* ***** ** * * ** ** * * * **

1UCU 349 YTADDGTSKTALNKLGGADGKTEVVSIGGKTYAASKAEGHNFKAQPDLAEAAATTTENPL

sfla 361 YTAADGTTKTAANQLGGVDGKTEVVTIDGKTYNASKAAGHDFKAQPELAEAAAKTTENPL

*** *** *** * *** ******* * **** **** ** ***** ****** ******

1UCU 409 QKIDAALAQVDTLRSDLGAVQNRFNSAITNLGNTVNNLTSVRSRIEDSDYATEVSNMSRA

sfla 421 QKIDAALAQVDALRSDLGAVQNRFNSAITNLGNTVNNLSEARSRIEDSDYATEVSNMSRA

*********** ************************** ******************

1UCU 469 QILQQAGTSVLAQANQVPQNVLSLLR (SEQ ID NO：840)

sfla 481 QILQQAGTSVLAQANQVPQNVLSLLR (SEQ ID NO：841)

**************************

Make to stimulate individual in the method for protein of protective immunological reaction can further comprise the step of nucleotide sequence that the proteic nucleotide sequence operability of code carrier is linked to the part of coding viral hemagglutination element.

" carrier (carrier) " is used for this paper and means, and can strengthen the molecule (for example, protein, peptide class) of the stimulation of protective immunological reaction.Carrier can be attached (for example,, chemical conjugation synthetic by recombinant technique, peptide or chemical reaction connect) through physics mode to compositions (protein portion of for example natural susceptible toxenia cell agglutinin), or with the said composition fusion.

The carrier that is used for the inventive method and compositions, can comprise that (for example) is at least a is selected from by tetanus toxoid (TT), the vibrio cholera toxoid, diphtheria toxoid (DT), the cross reactivity saltant (CRM) of diphtheria toxoid, the escherichia coli enterogenous endotoxin, the B subunit (LTB) of escherichia coli thermal instability enterogenous endotoxin, tobacco mosaic virus (TMV) (TMV) shell (coat) albumen, rabies virus (RV) peplos (envelope) albumen (glycoprotein), Elityran (Thy), heat shock protein HSP 60Kda, keyhole-limpet hemocyanin (keyhole limpet hemocyanin, KLH), early stage secretion antigen tuberculosis-6 (ESAT-6), exotoxin A, cholera toxoid (choleragenoid), the outer membrane protein complex of hepatitis B cAg and meningococcus (outer membrane protein complex, OMPC) member of the cohort of being formed (referring to, for example, how gloomyly permitted, R. wait the people, Prog Clin Biol Res 47:77-94 (1980); Perhaps how gloomy, people such as R., J Exp Med 152:361-76 (1980); Qiu, people such as C., Infect Immun 40:245-56 (1983); An Desen, P., Infect Immun 39:233-238 (1983); An Desen, people such as P., J Clin Invest76:52-59 (1985); Fragrant Brunswick, and people such as B.W. (Fenwick, B.W.), 54:583-586 (1986); Kui, people such as J.U., Infect Immun 56:2645-9 (1988); People such as Kui J.U., Infect Immun 56:2645-9 (1988); Kui, people such as J.U., Infect Immun 56:2645-9 (1988); Mu Lei, people such as K., Biol Chem380:277-283 (1999); Fragrant De Groot, people such as E., Vet Immunol Immunopathol 112:253-263 (2006); Qualifying Lan Nuofu, people such as D.M., vaccine 11:Suppl 1:S46-51 (1993)).

The exemplary carrier protein that is used for methods described herein and compositions can comprise at least a member who is selected from the cohort of being made up of SEQ ID NOS:788-795:

The cross reactivity saltant (CRM) of diphtheria toxoid comprises CRM197

GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKGFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKS(SEQ ID NO：788)

Tobacco mosaic virus (TMV) (TMV) coat protein

MMAYSIPTPSQLVYFTENYADYIPFVNRLINARSNSFQTQSGRDELREILIKSQVSVVSPISRFPAEPAYYIYLRDPSISTVYTALLQSTDTRNRVIEVENSTNVTTAEQLNAVRRTDDASTAIHNNLEQLLSLLTNGTGVFNRTSFESASGLTWLVTTTPRTA(SEQ ID NO：789)

Herba Medicaginis is inlayed the coat protein of virus (AMV)

MSSSQKKAGGKAGKPTKRSQNYAALRKAQLPKPPALKVPVAKPTNTILPQTGCVWQSLGTPLSLSSSNGLGARFLYSFLKDFAAPRILEEDLIFRMVFSITPSHAGSFCLTDDVTTEDGRAVAHGNPMQEFPHGAFHANEKFGFELVFTAPTHAGMQNQNFKHSYAVALCLDFDALPEGSRNPSYRFNEVWVERKAFPRAGPLRSLITVGLFDDADDLDRQ(SEQ ID NO：790)

The coat protein of Potyvirus X

MTTPANTTQATGSTTSTTTKTAGATPATTSGLFTIPDGEFFSTARAIVASNAVATNEDLSKIEAIWKDMKVPTDTMAQAAWDLVRHCADVGSSAQTEMIDTGPYSNGISRARLAAAIKEVCTLRQFCMKYAPVVWNWMLTNNSPPANWQAQGFKPEHKFAAFDFFNGVTNPAAIMPKEGLIRPPSEAEMNAAQTAAFVKITKARAQSNDFASLDAAVTRGRITGTTTAEAVVTLPPP(SEQ ID NO：791)

Be derived from Neisserial protein, for example

The I class 1 outer-membrane protein of meningococcus

MRKKLTALVLSALPLAAVADVSLYGEIKAGVEGRNYQLQLTEAQAANGGASGQVKVTKVTKAKSRIRTKISDFGSFIGFKGSEDLGEGLKAVWQLEQDVSVAGGGATQWGNRESFIGLAGEFGTLRAGRVANQFDDASQAIDPWDSNNDVASQLGIFKRHDDMPVSVRYDSPEFSGFSGSVQFVPAQNSKSAYKPAYWTTVNTGSATTTTFVPAVVGKPGSDVYYAGLNYKNGGFAGNYAFKYARHANVGRDAFELFLLGSGSDQAKGTDPLKNHQVHRLTGGYEEGGLNLALAAQLDLSENGDKTKNSTTEIAATASYRFGNAVPRISYAHGFDFIERGKKGENTSYDQIIAGVDYDFSKRTSAIVSGAWLKRNTGIGNYTQINAASVGLRHKF(SEQ IDNO：792)

Main pili subunit protein I type (pilin (Fimbrillin))

MVLKTSNSNRAFGVGDDESKVAKLTVMVYNGEQQEAIKSAENATKVEDIKCSAGQRTLVVMANTGAMELVGKTLAEVKALTTELTAENQEAAGLIMTAEPKTIVLKAGKNYIGYSGTGEGNHIENDPLKIKRVHARMAFTEIKVQMSAAYDNIYTFVPEKIYGLIAKKQSNLFGATLVNADANYLTGSLTTFNGAYTPANYANVPWLSRNYVAPAADAPQGFYVLENDYSANGGTIHPTILCVYGKLQKNGADLAGADLAAAQAANWVDAEGKTYYPVLVNFNSNNYTYDSNYTPKNKIERNHKYDIKLTITGPGTNNPENPITESAHLNVQCTVAEWVLVGQNATW(SEQ ID NO：793)

Fermentation mycoplasmas (Mycoplasma fermentans) macrophage activation lipopeptid (MALP-2)

MKKSKKILLGLSPIAAVLPAVAVSCGNNDESNISFKEKDISKYTTTNANGKQVVKNAELLKLKPVLITDEGKIDDKSFNQSAFEALKAINKQTGIEINSVEPSSNFESAYNSALSAGHKIWVLNGFKHQQSIKQYIDAHREELERNQIKIIGIDFDIETEYKWFYSLQFNIKESAFTTGYAIASWLSEQDESKRVVASFGVGAFPGVTTFNEGFAKGILYYNQKHKSSKIYHTSPVKLDSGFTAGEKMNTVINNVLSSTPADVKYNPHVILSVAGPATFETVRLANKGQYVIGVDSDQGMIQDKDRILTSVLKHIKQAVYETLLDLILEKEEGYKPYVVKDKKADKKWSHFGTQKEKWIGVAENHFSNTEEQAKINNKIKEAIKMFKELPEDFVKYINSDKALKDGNKIDNVSERLEAIISAINKAAK(SEQ ID NO：794)

The P19 protein of mycobacterium tuberculosis

ATTLPVQRHPRSLFPEFSELFAAFPSFAGLRPTFDTRLMRLEDEMKEGRYEVRAELPGVDPDKDVDIMVRDGQLTIKAERTEQKDFDGRSEFAYGSFVRTVSLPVGADEDDIKATYDKGILTVSVAVSEGKPTEKHIQIRSTN(SEQ ID NO：795)

The present composition can further comprise at least a adjuvant.Adjuvant contain can strengthen antagonism itself have difference immunoreactive dose of immunogenic material (referring to, for example, immunological method handbook (Immunology Methods Manual), volume 2, I. Li Fukewei Si is compiling, and institute publishes (AcademicPress), Santiago, CA, 1997, ch.13).The immunological method handbook can buy be four the volume serial books (product code name Z37,435-0); CD-ROM CD book (product code name Z37,436-9); Or the two is complete (product code name Z37,437-7).Adjuvant can be the mixture of (for example) natural or synthetic compound, and it can further strengthen this proteinic immunoreation when with the present composition (for example protein of the stimulation protective immunological reaction that makes by methods described herein) administration.The compositions that further comprises adjuvant can be passed through (for example) irritation cell and/or humoral response (that is the protective effect antagonist of release disease is made), and further strengthens the protective immunological reaction by the present composition stimulated.Adjuvant can be via promoting protein adsorption with location, extension or increase protein release, macrophage activation effect, reach T and B cytositimulation and the execution effect.The adjuvant that is used for methods described herein and compositions can be inorganic salts, fat liquor, mycobacteria product, Saponin, synthetic product and cytokine.Adjuvant can be attached (for example, by recombinant technique, synthetic or chemical reaction connects by peptide) through physics mode to compositions described herein, or with the said composition fusion.

In another embodiment, the present invention is a kind of method of protein that stimulates individual interior protective immunological reaction of making, it comprises isolates a proteinic part from the natural viral hemagglutinin, form protein portion thus, wherein this protein portion comprises at least a portion and at least a at least a portion that makes ball head in fact still possess the secondary structure of its tertiary structure of ball head, and wherein this protein portion lacks film fusion function territory, strides film functional domain and kytoplasm functional domain; The nucleotide sequence commentaries on classics of this protein portion of coding is infected to the eucaryon host cell, and wherein this eukaryotic host cell is not pichia pastoris phaff (Pichia pastoris) eukaryotic host cell; Reach the proteinic step of cultivating this eukaryotic host cell and making the individual interior protective immunological reaction of stimulation thus.In another embodiment, this protein portion can further lack signal sequence.In another embodiment, this protein portion can further comprise the sialic acid binding site.

In another embodiment, the present invention is a kind of method of protein that stimulates individual interior protective immunological reaction of making, it comprises isolates a proteinic part from the natural viral hemagglutinin, form protein portion thus, wherein this protein portion comprises at least a portion and at least a at least a portion that makes ball head in fact still possess the secondary structure of its tertiary structure of ball head, and wherein this protein portion lacks film fusion function territory, strides film functional domain and kytoplasm functional domain; The nucleotide sequence commentaries on classics of this protein portion of coding is infected to the eucaryon host cell, and wherein this eukaryotic host cell is not the insecticide eukaryotic host cell; Reach the proteinic step of cultivating this eukaryotic host cell and making the individual interior protective immunological reaction of stimulation thus.

In an embodiment, the present invention is a kind of method of protein that stimulates individual interior protective immunological reaction of making, it comprises isolates a proteinic part from the natural viral hemagglutinin, form protein portion thus, wherein this protein portion comprises at least a portion and at least a at least a portion that makes ball head in fact still possess the secondary structure of its tertiary structure of ball head, and wherein this protein portion lacks film fusion function territory, strides film functional domain and kytoplasm functional domain; The nucleotide sequence commentaries on classics of this protein portion of coding is infected to the eucaryon host cell, and wherein this eukaryotic host cell is not the insect host cell through stable conversion; Reach the proteinic step of cultivating this eukaryotic host cell and making the individual interior protective immunological reaction of stimulation thus.

In another embodiment, the present invention is a kind of method of protein that stimulates individual interior protective immunological reaction of making, it comprises isolates a proteinic part from the natural viral hemagglutinin, form protein portion thus, wherein this protein portion comprises at least a portion and at least a at least a portion that makes ball head in fact still possess the secondary structure of its tertiary structure of ball head, and wherein this protein portion lacks film fusion function territory, strides film functional domain and kytoplasm functional domain; The nucleotide sequence commentaries on classics of this protein portion of coding is infected to insect cell host cell (for example, the baculovirus insect host cell is as Sf9 or High5 cell); Reach the proteinic step of cultivating this eukaryotic host cell and making the individual interior protective immunological reaction of stimulation thus.

Make the method for protein that stimulates individual interior protective immunological reaction, can further comprise the step that lacks the saccharifying site at least one nucleotide sequence that is present in this protein portion of encoding.Saccharifying site through disappearance can comprise N-saccharifying site.

The eukaryotic host cell that is used for the inventive method, (for example can comprise Saccharomyces eukaryotic host cell, insecticide eukaryotic host cell, at least a being selected from by insect cell through baculovirus infection, as meadow armyworm (Spodoptera frugiperda) (Sf9) or (High5) cell of cabbage looper (Trichhoplusia ni); The member of the cohort of being formed with fruit bat insect cell such as Dm12 cell), fungus eukaryotic host cell, parasite eukaryotic host cell (for example leishmania (Leishmania tarentolae) eukaryotic host cell), Chinese hamster ovary celI, yeast cell (for example Pichia sp.) and Kluyveromyces lactis (Kluyveromyceslactis) host cell.

Suitable eukaryotic host cell and carrier also can comprise plant cell (for example, Fructus Lycopersici esculenti; Chloroplast; Single-with the dicotyledon cell; Arabic mustard (Arabidopsis thaliana); Fructus Hordei Vulgaris; Corn; Rhizoma Solani tuber osi is Solanum tuberosum for example; Radix Dauci Sativae is Radix Dauci Sativae (Daucus carota L) for example.; And Nicotiana tabacum L. for example Nicotiana tabacum, Nicotiana benthamiana (gill this, people such as M., PlantBiotechnol be (2005) J.3:613-20; Seat, people such as D.M., Colloids Surf B Biointerfaces (2006); Huang, people such as Z., vaccine 19:2163-71 (2001); Kant watt, people such as A., virusology 308:207-15 (2003); Ma Kuite-Bron, people such as E., Plant Mol Biol 51:459-69 (2003); Su Daxiena, people such as M.R., Plant Biotechnol is (2006) J.4:551-9; Method Sani, people such as A., VirusRes, 120:91-6 (2006); Karma adds people such as reaching S., Expert Rev Vaccines 5:839-49 (2006); Dell V waits the people, Infect Immun.73:8266-74 (2005); Open, people such as X., Plant Biotechnol is (2006) J.4:419-32).

The protein that makes by the inventive method, and compositions of the present invention can utilize well-known process (for example, gel chromatography art, cation-exchange chromatography art, SDS-PAGE) to carry out purification and characterization.

For large-scale production, can use fermentation technique as described herein.Other exemplary fermentation technique can comprise a kind of circulation that has proposed, and it can be from going into the culture (as described herein) of the MRBR culture medium of 6L through inoculation, remains in about 30 ℃, about pH 7 and controls DO extremely greater than about 30%.Can exhaust the back at least about 30 minutes at glucose then, begin 6 liters of feedings.6 liters of feeding culture medium that proposed when the MRBR culture medium with 6L makes up, can provide the escherichia coli growth conditions needed based on about 52% growth carbon utilisation rate.Feeding can or can not comprise IPTG.Can induce criticizing part with 2mM IPTG at least (with the bolus form, begin to start import soon after producing in feeding).Rate of feeding can start from per hour every liter of biological reaction tank volume of about 20mL feeding, can accept more glucosan and not cause the cumulative ability of glucose based on culture afterwards, and increase in time.When feeding is finished, can collect culture.6 liters of feeding culture medium (about pH 6.0) can comprise glucose 180g/L; KH ₂PO ₄2g/L; NaH ₂PO ₄(H ₂O) 4g/L; (NH ₄) ₂HPO ₄12g/L; (NH ₄) ₂HSO ₄4g/L; DL-alanine 40g/L; Citric acid 4g/L; MgSO ₄(7H ₂0) 5.5g/L; Trace meter 6mL; CaCl ₂2.5g/L; FeSO ₄7H ₂O 1g/L.

Cytoclasis in extensive manufacturing and clarificationization can comprise from the resuspending buffer and remove TritonX-100; By with 50mM Tris, 25mM NaCl, 8M carbamide, about pH 8 adds to lysate and makes the insoluble matter dissolving; Add PEI (poly-ethamine) and then carry out centrifugal removing, filter to remove nucleic acid and/or help with one or more buffer; Add flocculating agent for example Aerosil 380, Aerosil 200, AlkoxideAlu C and Celpur; And then remove to help filtration by centrifugal.The cation-exchange chromatography art can comprise uses the processing resin, and increase it and contain 8M carbamide and the degeneration endotoxin removal step of about 2% Triton X-100 at the most at the most, and the step gradient eluting.The step gradient can comprise about 100 to about 200mMNaCl.

In another embodiment, the present invention is a kind of method that stimulates individual interior protective immunological reaction, it comprises and comprises that with a kind of the proteinic compositions administration that makes by the method that comprises following steps gives this individual step: a proteinic part is separated from the natural viral hemagglutinin, form protein portion thus, wherein this protein portion comprises at least a portion and at least a at least a portion that makes ball head in fact still possess the secondary structure of its tertiary structure of ball head, and wherein this protein portion lacks film fusion function territory, stride film functional domain and kytoplasm functional domain; The nucleotide sequence of this part of coding is converted in the prokaryotic host cell; Reach the protein of cultivating this prokaryotic host cell and making the individual interior protective immunological reaction of stimulation thus.

In another embodiment; the present invention is a kind of method that stimulates individual interior protective immunity; it comprises and comprises that with a kind of the compositions administration of the protein portion that derives from the natural viral hemagglutinin gives this individual step; wherein this protein portion comprises at least a portion and a kind of at least a portion that makes ball head in fact still possess the secondary structure of its tertiary structure of ball head, and wherein this protein portion lacks film fusion function territory, strides film functional domain and kytoplasm functional domain.

In other embodiment, the present invention is a kind of method of making the viral hemagglutination fibroin that stimulates individual interior protective immunological reaction, it comprises isolates a proteinic part from the natural viral hemagglutinin, form protein portion thus, wherein this protein portion comprises at least a portion and at least a at least a portion that makes ball head in fact still possess the secondary structure of its tertiary structure of ball head, and wherein this protein portion lacks film fusion function territory, strides film functional domain and kytoplasm functional domain; The nucleotide sequence commentaries on classics of this protein portion of coding is infected to the eucaryon host cell, and wherein this eukaryotic host cell is not the pichia pastoris phaff eukaryotic host cell; Reach the proteinic step of cultivating this eukaryotic host cell and making the individual interior protective immunological reaction of this stimulation thus.

In another embodiment, the present invention is a kind of method of making the viral hemagglutination fibroin that stimulates individual interior protective immunological reaction, it comprises isolates a proteinic part from the natural viral hemagglutinin, form protein portion thus, wherein this protein portion comprises at least a portion and at least a at least a portion that makes ball head in fact still possess the secondary structure of its tertiary structure of ball head, and wherein this protein portion lacks film fusion function territory, strides film functional domain and kytoplasm functional domain; The nucleotide sequence commentaries on classics of this protein portion of coding is infected to the eucaryon host cell, and wherein this eukaryotic host cell is not the Drosophila melanogaster eukaryotic host cell; Reach the proteinic step of cultivating this eukaryotic host cell and making the individual interior protective immunological reaction of this stimulation thus.

In another embodiment, the present invention is a kind of method of making the viral hemagglutination fibroin that stimulates individual interior protective immunological reaction, and it comprises the nucleotide sequence that prokaryotic host cell is striden the viral hemagglutination element of film functional domain and kytoplasm functional domain with at least a shortage of encoding and transforms; Reach the proteinic step of cultivating this prokaryotic host cell and making the individual interior protective immunological reaction of this stimulation thus.

In the other embodiment of the present invention for a kind of stimulate individual in the method for protection immunity, it comprises this individual step is given in a kind of proteinic compositions administration that comprises that the method by following steps makes: the nucleotide sequence that prokaryotic host cell is striden the viral hemagglutination element of film functional domain and kytoplasm functional domain with at least a shortage of encoding transforms; Reach the protein of cultivating this prokaryotic host cell and making the individual interior protective immunological reaction of this stimulation thus.

In another embodiment; the present invention is a kind of method that stimulates individual interior protection immunity; it comprises and comprises that with a kind of having a kind of shortage strides the proteinic compositions administration of the viral hemagglutination element of film functional domain and kytoplasm functional domain and give this individual step, and wherein this protein expression is in prokaryotic cell.

In another embodiment, the present invention is the compositions of the protein portion of at least a portion of a kind of molecule pattern that comprises at least a pathogen-connection and natural viral hemagglutinin, wherein the protein portion of this natural viral hemagglutinin comprises at least a portion and at least a at least a portion that makes ball head in fact still possess the secondary structure of its tertiary structure of ball head, and wherein the protein portion of this natural viral hemagglutinin lacks film fusion function territory, strides film functional domain and kytoplasm functional domain.

Pathogen-connection molecule pattern (PAMP), for example flagellin or antibacterial fat egg, meaning a class exists in the microorganism, when with a pattern recognition receptor (pattern recognition receptor, PRR) in conjunction with the time, can cause the molecule (for example, protein, peptide, saccharide, lipid, lipoprotein, nucleic acid) of natural immunity reaction.PRR can be clock sample receptor (TLR).

TLR has been made maximum classes for the feature that is expressed in antigen-the represent pattern recognition receptor (PRR) on the sexual cell (APC).APC utilizes TLR to reconnoitre microenvironment, and by detect the signal of pathogenic infection in conjunction with the connection part (PAMP) of TLR.PAMP and TLR interact and can cause natural immunity reaction, first line defence that it is invaded for the enantiopathy substance, and it shows as and disengages cytokine, chemotactic factor and other inflammation mediation material; Convene the phagocyte sexual cell; And cause common zest developed by molecule, and antigen effectively processed and represent the important molecule mechanism of giving the T-cell.

PAMP and TLR combine the active natural immunization route.The target cell can impel zest molecule and combination mainly organize the antigen of compatible complex molecule to be showed in cell surface (referring to Figure 40) altogether.Compositions of the present invention, fused protein or polypeptide can comprise and the bonded PAMP of TLR (for example TLR5) (for example flagellin), promote the APC differentiation with ripe, comprise the manufacturing and the displaying (referring to Figure 40) of common zest signal.Compositions can be by it with the reciprocal action of TLR and by internalization, and processes and produce the antigenic peptide class through lysosomal pathway, and it is by to be showed on the surface with the bonded mode of the compatible complex molecule of main tissue.

Compositions of the present invention, fused protein or polypeptide utilization cause the pathogen-connection molecule pattern (TLR agonist) that causes following cell incident: be situated between plain and chemokine secretion of zest developed by molecule, important cells altogether; And antigen effectively processed and represent give the T-cell.As mentioned above, TLR identification PAMP comprises bacteria cell wall composition (for example, bacterial lipoprotein and lipopolysaccharide), contains not the DNA of bacteria sequence and the bacterial flagellin of methylated CpG residue.TLR act as initiation of natural immunity reaction, with gate management and control person (the appropriate husband of U.S. seat, people such as R., the Cold Springs Harb.Symp.Quant.Biol.64:429 (1999) of adaptive immunity reaction; Pai Saer, people such as C.R., Semin, Immunol 16:23 (2004); The appropriate husband of U.S. seat, people such as R., Nature 388:394 (1997); Ba Dun, people such as G.M., Curr.Opin.Immunol14:380 (2002); Class's moral lek, people such as A., J.Exp.Med.195:F19 (2002)).

The as above dissertator of institute, the immunization route that activation is used for the present composition, fused protein and polypeptide that combines of PAMP and TLR, its can be used for stimulating individual in immune system.Compositions of the present invention, fused protein and polypeptide at a certain antigen (for example can cause, virus protein such as influenza virus) immunoreation, and cause the signal pipeline of individual natural and adaptive immune system, and stimulate the individual immunity system thus.The stimulation of individual immunity system can prevent the infection that caused by antigen or virus (for example influenza virus), and treats this individuality thus, or prevention is individual that disease, disease and (possibly) death takes place.

In an other embodiment, the present invention comprises it it is the compositions of the flagellin composition of the part of flagellin at least for a kind of, wherein this flagellin composition comprises at least one cysteine residues, and makes this flagellin composition activation clock sample receptor 5 thus.

" flagellin composition " is used for this paper and means, at least a portion of complete flagellin.

" activation " means this composition (for example, flagellin composition or clock sample receptor stimulating agent are formed) when about clock sample receptor (TLR) can stimulate the reaction of correlating with TLR.For example, bacterial flagellin stimulates TLR5 and host's inflammatory response (Smith, people such as K.D., natural immunity (Nature Immunology) 4:1247-1253 (2003)).Antibacterial lipopeptid activation TLR1; Pam3Cys, Pam2Cys activate TLR2; DsRNA activates TLR3; LBS (LPS-is conjugated protein) and LPS (lipopolysaccharide) activation TLR4; Imidazoquinolie (anti--virus compound and ssRNA) activation TLR7; And DNA of bacteria (CpG DNA) activation TLR9.TLR1 and TLR6 need carry out different first double focusingization with TLR2, with recognition ligand (for example TLR agonist, TLR antagonist).TLR1/2 is by three acyl group lipoproteins (or lipopeptid, for example Pam3Cys) activation, and TLR6/2 is by diacyl lipid albumen (for example Pam2Cys) activation, exists though have some intersection identification.Except native ligand, the synthesis type micromolecule comprises that imidazoquinolie (specific class of couple TLR7 or TLR8 tool wherein arranged) can stimulate TLR7 and TLR8.The synthesis type LPS analog that can activate TLR4 is also arranged, for example monophosphoryl lipid A [MPL].

The activation of TLR can cause seeing through the citation that MyD88 and NF-κ B carry out.More existing evidences point out that different TLR induce different immune results.For example, people such as conspicuous Fei perhaps, Infect Immun69:1477-1482 (2001) and Lei Dengren, J Biol Chem 276:37692-37699 (2001) confirm that TLR2 activates different gene performance kenels with TLR4 in dendritic cell.The general Lan Dengren that gets, J Immunol167:5067-5076 (2001) proves, the gene performance kenel of these differences can be reappeared on the protein level in antigen-specific reaction, when the lipopolysaccharide that use to see through TLR2 or TLR4 citation is guided this reaction (TLR4 tends to follow and enriches the excretory class Th1 reaction of IFN γ, and TLR2 tends to follow the Th2-that enriches IL-5, IL-10 and IL-13 and hang down IFN γ level to react).The result who sees through different TLR citations has a variety of.

The activation of TLR can cause, and can increase and detected effector lymphocyte is active for example by measuring IFN gamma-secretase CD8+ cell (for example cytotoxic T-cytoactive); Can increase by for example ELISA, virus neutralization and the detected antibody response of flow cytometry technology and (be permitted Nai Er, people such as M., NatImmunol 2:947 (2001); Ya Liebolu, people such as L., Nat Med 8:878 (2002); Pai Saer, people such as C., science (Science) 299:1033 (2003); Take the ripple Brittany, people such as G., Nat Immunol 6:769 (2005); And Ai Bo Qwest, people such as S.E., J Immunol 175:3882 (2005)).

(wherein this flagellin composition comprises at least one cysteine residues for the compositions of the flagellin composition of at least a portion of flagellin to comprise it, and make this flagellin composition activate clock sample receptor 5 thus), can further comprise at least a being selected from by clock sample receptor 1 agonist, clock sample receptor 2 agonists (for example, Pam3Cys, Pam2Cys), clock sample receptor 3 agonist, clock sample receptor 4 agonist, clock sample receptor 6 agonist, clock sample receptor 7 agonist, clock sample receptor 8 agonist, clock sample receptor 9 agonist, clock sample acceptor 10 agonist, at least a portion of the member of the cohort that clock sample receptor 11 agonist and clock sample receptor 12 agonist are formed.

Be used in compositions clock sample receptor 1 agonist of (for example be included as the compositions of the flagellin composition that is a flagellin part at least, wherein this flagellin composition comprises at least one cysteine residues and this flagellin composition activation clock sample receptor 5), clock sample receptor 2 agonists, clock sample receptor 3 agonist, clock sample receptor 4 agonist, clock sample receptor 6 agonist, clock sample receptor 7 agonist, clock sample receptor 8 agonist, clock sample receptor 9 agonist, clock sample acceptor 10 agonist, clock sample receptor 11 agonist and clock sample receptor 12 agonist can further comprise the cysteine residues that at least one is extra.

In an embodiment, at least one cysteine residues is the amino acid residue that replaces at least one natural flagellin aminoacid sequence that has the flagellin composition.

The cysteine residues that replaces the amino acid residue at least one natural flagellin aminoacid sequence that has the flagellin composition can be away from least one aminoacid of clock sample receptor 5 recognition sites of flagellin composition." clock sample receptor 5 recognition sites " mean and the part of TLR5 interaction with the TLR5 part (for example, TLR5 agonist) of mediated cell reaction." clock sample receptor 5 recognition sites " also are called " clock sample receptor 5 activation sites " and reach " clock sample receptor 5 mobilizing function territories ".

Equally, " clock sample receptor recognition site " means, and TLR discrete with it interacts with the part of the TLR part (for example, TLR agonist) of mediated cell reaction." clock sample receptor recognition site " also is called " clock sample receptor activation site " and reaches " clock sample receptor activation functional domain ".

The cysteine residues that replaces the amino acid residue at least one natural flagellin aminoacid sequence that has the flagellin composition also can relating to and clock sample receptor 5 bonded at least one aminoacid away from the flagellin composition.

In another embodiment, the flagellin composition comprises at least and the bonded natural flagellin aminoacid sequence part of cysteine residues.

Be used for can be at least a amino-end amino acid that is selected from by flagellin composition or clock sample receptor stimulating agent with the bonded cysteine residues of at least a portion of natural flagellin aminoacid sequence (this paper also is called " cysteine that is added "), and member's (being used for being used in combination with it) of the cohort formed of the carboxyl-end amino acid of flagellin composition or clock sample receptor stimulating agent.Be used for the bonded cysteine residues of at least a portion of natural flagellin aminoacid sequence can be away from least one aminoacid of clock sample receptor 5 recognition sites of flagellin composition, or away from least one aminoacid of the clock sample receptor recognition site of clock sample receptor stimulating agent.

Be used for the bonded cysteine residues of natural flagellin aminoacid sequence part can be away from the relating to and clock sample receptor 5 bonded at least one aminoacid of flagellin composition, or away from least one aminoacid with clock sample receptors bind of relating to of clock sample receptor stimulating agent.In another embodiment, the flagellin composition lacks at least a portion of natural flagellin aminoacid sequence part hinge region.

Comprise it and be the flagellin composition of the part of flagellin at least, wherein this flagellin composition comprises at least one cysteine residues and makes this flagellin composition activate the compositions of clock sample receptor 5 thus, the part that can further comprise at least a antigen (for example, influenza antigen such as influenza virus A, B or C antigen).

This antigen can be hydrophobicity antigen in fact, for example the ripe shearing site of HA.Ripe shearing site antigen can be at least a NOS:530 by SEQ ID that is selected from, 532,533,534,535,600,601,602,603 and NVPEKQTRGIFGAIAGFIE (H3) (SEQ ID NO:796), NIPSIQSRGLFGAIAGFIE (H1) (SEQ ID NO:797), PAKLLKERGFFGAIAGFLE (FLU B) (SEQ ID NO:798), RERRRKKRGLFGAIAGFIE (H5) (SEQ ID NO:799), RGLXGAIAGFIE (SEQ ID NO:821), RGLXGAIAGFIE (the member of the cohort that SEQ ID NO:822 is formed.

" hydrophobicity in fact " is used for this paper and means, and this antigen has limited dissolubility in aqueous solution or environment.Peptide or proteinic hydrophobic property can and compare by (for example) Kate-Du Lier hydrophobicity scale (Kate, people such as J., Mol.Biol.157:105-132 (1982)) mensuration, and it specifies a numerical value according to relative hydrophobicity for each 20 seed amino acid.On the occasion of being expressed as hydrophobic amino acid.Negative value is expressed as hydrophilic amino acid.For a kind of indivedual peptides or polypeptide, these numerical value average (by will be for this polypeptide or proteinic each amino acid whose indivedual hydrophobicity numerical value addition, and with total value divided by this polypeptide or proteinic amino acid number and calculate), overall hydrophobic index can be provided, be called " hydrophobic grand average ", also be called ＂ GRAVY ＂.The GRAVY value is hydrophobicity in fact greater than this protein of zero representation or peptide.The GRAVY value is hydrophilic in fact less than this protein of zero representation or peptide.According to Kate-Du Lier scale (Kate, people such as J., Mol.Biol.157:105-132 (1982)), as follows for indivedual hydrophobicity numerical value of 20 kinds of natural amino acids:

Alanine 1.8

Arginine-4.5

Agedoite-3.5

Aspartic acid-3.5

Cysteine 2.5

Glutamine-3.5

Glutamic acid-3.5

Glycine-0.4

Histidine-3.2

Isoleucine 4.5

Leucine 3.8

Lysine-3.9

Methionine 1.9

Phenylalanine 2.8

Proline-1.6

Serine-0.8

Threonine-0.7

Tryptophan-0.9

Tyrosine-1.3

Valine 4.2

The hydrophobicity that is used for the antigen (for example protein or peptide antigen) of the present composition and method can be by based on the hydrophobicity numerical value of aforementioned Kate-Du Lier (Kyte-Doolittle) scale, calculates its GRAVY mark and measure.For example, it is designated as the GRAVY mark of hydrophobic peptide in fact, is by calculating for indivedual amino acid whose hydrophobicity numerical value (as the above-mentioned) addition of following ripe shearing site amino acid peptide:

The ripe position of shearing GRAVY

NVPEKQTRGIFGAIAGFIE(A/H3N2) (SEQ ID NO：800) 0.053

NVPQIESRGLFGAIAGFIE(A/H2N1) (SEQ ID NO：801) 0.453

NIPSIQSRGLFGAIAGFIE(A/H1N1) (SEQ ID NO：802) 0.611

RGLFGAIAGFIE (influenza virus A conserved region) (SEQ ID NO:803) 1.067

PAKLLKERGFFGAIAGFLE (influenza virus B) (SEQ ID NO:804) 0.400

RGFFGAIAGFLE (influenza virus B conserved region) (SEQ ID NO:805) 0.925

Equally, calculate it for following peptide and be designated as the GRAVY mark of hydrophobic peptide in fact:

Aminoacid sequence GRAVY

Human influenza virus M2e (SEQ ID NO:806)

SLLTEVETPIRNEWGSRSNDSSDP -1.129

Vietnam influenza m 2 e (SEQ ID NO:807)

GSGAG SLLTEVETPTRNEWECRCSDSSDP -0.907

Mao flu virus M2e (SEQ ID NO:808)

GSGAGSLLTEVETLTRNGWGCRCSDSSDP -0.507

West nile virus E peptide 001 (SEQ ID NO:809)

LTSGHLKCRVKMEKLQLKGT -0.475

Dengue fever 2E peptide (SEQ ID NO:810)

EAEPPFGDSYIIIGVEPGQLKLNWFKK -0.333

BCRABLwt peptide (SEQ ID NO:811)

SLLTEVETPIRNEWGSRSNDSSDP -1.129

Antigen can be any molecule (for example, protein, peptide, glycoprotein, glycopeptide, carbohydrate, lipid, lipopeptid, polysaccharide) that can be discerned by immune composition, no matter and whether it can cause immune activation.Antigen can be the fragment or the part of native antigen, or is the synthesis type molecule of the part of simulation native antigen or native antigen.

Antigen can be antigenic virus." antigenic virus " is used for this paper and means, when any part that is used in combination or can produces immunoreactive virus (for example influenza virus, banzi virus) with TLR agonist (for example flagellin, Pam2Cys, Pam3Cys) under no TLR agonist exists in individual.Virus antigen can be a part or the fragment of natural viral, or is the synthesis type molecule of simulative virus, for example produces immunoreactive reorganization or synthesis type protein (for example, influenza virus, banzi virus), peptide, lipid, carbohydrate in individual.Influenza antigen can comprise at least a member who is selected from the cohort of being made up of influenza virus A antigen, influenza virus B antigen and influenza virus C antigen.Influenza antigen can be influenza virus conformability memebrane protein, for example HA, or HA protein portion, for example HA1-1 and HA1-2.

Antigen can be the proteic at least a portion of influenza virus substrate 2 (M2), comprises at least a portion of the outer functional domain of born of the same parents of influenza m 2 protein (M2e).

Stromatin 2 (M2 or M2 albumen) is the proton selectivity conformability film ionophorous protein of influenza virus A.M2 is still generally expressed by virion is not enough by the abundant cytoplasmic membrane that is expressed in the cell that is infected by the virus.For example, the part of the M2 sequence of influenza virus A is SEQ ID NO:508, and it is to be encoded by SEQID NO:509.The proteic native form of M2 is with first tetramer (that is, four identical M2 protein moleculars that connect with disulfide bond).Each unit is for passing through two spirals that disulfide bond is stable.M2 is activated by low pH value.Form by three functional domains with each the M2 protein molecular in first tetramer: outer or N (amino)-terminal functional domain (for example, the SEQ ID NO:510 of 24 aminoacid; Also be called " human consensus sequence " in this paper), it is to be encoded by SEQ ID NO:511; 19 hydrophobic amino acids are striden diaphragm area; And 54 aminoacid are interior or C (carboxyl)-terminal functional domain.M2 albumen can be with influenza virus sub-strain (for example, the H1 of influenza virus A and H5 hypotype) and influenza virus source (for example Puerto Rico, Thailand, New York, Hong Kong) and change,, reach described in PCT/US2005/046662 (WO2006/069262) in shown in the proteic exemplary amino-end sequence of M2 (SEQ ID NOS:544-556 and 570-578) as for example.

M2 albumen is being played the part of important role in the life cycle of A type influenza virus.Enter in the virion because of it allows ion, reduce the inner pH value of virus, cause viroplast albumen M1 to dissociate, so have importance sloughing shell (uncoating) stage from nucleoglucoprotein RNP.As a result, virus coat removes, and the content of virus just disengages from endosome, and enters in the Cytoplasm of host cell to infect.

The function of M2 channel can be subjected to antiviral drugs, and for example amantadine (mantadine) suppresses with rimantadine (rimantadine), and it prevents the viral infection host cell.This type of antiviral drugs is striden the film district in conjunction with M2 is proteic usually, and the ion channel that blocking-up is produced by M2 albumen on the space, and prevents that proton from entering and make virion slough shell.

The M2 albumen that is used for the present composition and method can comprise at least a portion of SEQ ID NO:510 (by SEQ IDNO:511 coding) or at least a portion of SEQ ID NO:544 (by SEQ ID NO:603 coding).M2 albumen can further comprise at least a being selected from by SEQ ID NO:512, SEQID NO:516, SEQ ID NO:531; SEQ ID NO:536 (Flu A H5N1 M2e, 2004 Vietnam's separated strains have serine displacement cysteine); SEQ ID NO:537 (Flu A H5N1 M2e, 2004 Vietnam's separated strains); SEQ ID NO:538 (Flu A H5N1 M2e, Hong Kong 97 separated strains have serine displacement cysteine); SEQ ID NO:539 (FluA H5N1 M2e, Hong Kong 97 separated strains); SEQ IDNO:540 (Flu A H7N2 M2e chicken/New York 95 separated strains have serine displacement cysteine); SEQID NO:541 (Flu A H7N2 M2e, chicken/New York 95 separated strains); SEQ ID NO:542 (Flu A H9N2M2e, Hong Kong 99 separated strains have serine displacement cysteine); And the member of the cohort formed of SEQ ID NO:543 (Flu A, Hong Kong 99 separated strains).Some exists the specific cysteine residues in the native sequences of the proteic at least a portion of M2, for example amino acid/11 6 and 18 of SEQ ID NO:537; SEQ IDNOS:539,541 and 543 amino acid/11 7 and 19 are with serine (referring to, SEQ ID NOS:538,540,542 and 544 respectively) through displacement.

Be included as at least a portion of flagellin the flagellin composition (wherein this flagellin composition comprise at least one cysteine residues and thus the activation of this flagellin composition (for example further comprise antigen, ripe shearing site wins peptide, the HA protein portion, HA1-1 for example, HA1-2) compositions clock sample receptor 5) can with another be at least flagellin part the flagellin composition (wherein this flagellin composition comprise at least one cysteine residues and thus this flagellin composition activation further comprise different, clear and definite antigen (for example, clock sample receptor 5) or TLR agonist and antigen (for example, STF2.HA1-1 M2e), STF2.HA1-2, STF2 Δ .HA1-1, STF2 Δ .HA1-2, STF2.M2e, STF2.4xM2e, STF2 Δ .M2e, STF2 Δ .4xM2e) fusion rotein is together thrown and is given or mix.

The antigen that is contained in compositions of the present invention and is used for method of the present invention can be the microorganism related antigen, for example the antigenic at least a portion of pathogen (as virus, fungus or antibacterial).It is relevant that antigen also can be microorganism, and with the antigen of other disease association, for example with the antigen of allergy and cancer connection.Antigen also can be antigenic at least a portion of antibacterial, virus, fungus, yeast, protozoacide, metazoa, tumor, malignant cell, plant cell, zooblast, hormone and amyloid-β peptide.Antigen can be a kind of peptide, polypeptide, lipoprotein, glycoprotein and mucoprotein.

The antigen that is contained in compositions of the present invention and is used for method of the present invention can be the relevant antigen of pathogen." antigen that pathogen is relevant " is used for this paper and means any molecule (for example, protein, peptide, carbohydrate, lipoprotein, polysaccharide) that correlates with pathogen.The antigen that exemplary pathogen is relevant comprises (for example) poxvirus, fowlpox virus, the turkey influenza virus, bovine leukemia virus, feline leukaemia virus, bird flu virus, the chicken pneumonitis virus, the dog small virus, equine influenza virus, FHV, new castle disease virus (NDV), chicken/Binzhou/1/83 influenza virus, infectious bronchitis virus, dengue virus, Measles virus, rubella virus, pseudorabies, Yi Sitan-Ba Er (Epstein-Barr) virus, HIV, SIV, EHV, BHV, HCMV, Hantaan, clostridium tetani (C.tetani), parotitis, Morbillivirus, herpes simplex virus the 1st type, herpes simplex virus the 2nd type, the huge sexually transmitted disease (STD) poison of human cell, hepatitis A virus, hepatitis virus B, hepatitis C virus, the E Hepatitis virus, respiratory tract amalgamation virus, human papillomavirus, influenza virus, Salmonella, Neisseria, burgdorferi, chlamydia, the Bo Deshi bacillus, Plasmodium (for example Plasmodium falciparum (Plasmodium falciparum) and Plasmodium vivax), toxoplasma, Cryptococcus, Streptococcus, staphylococcus, Haemophilus spp, diphtheria, tetanus, pertussis, Colibacter, Candida, aspergillus, Endamoeba, Giardia and trypanosoma.The antigen that is contained in the present composition and is used for method of the present invention can comprise bacterial capsule antigen.Exemplary bacterial capsule antigen comprises that (for example) is at least a and is selected from by B group streptococcus (comprising streptococcus agalactiae) capsular polysaccharide Ia, Ib, Ia/c, II, III, IV, V and VI type; Pneumonia group streptococcus capsular polysaccharide 1,2,3,4,5,6A, 6B, 7F, 9N, 9V, 10A, 11A, 12F, 14,15B, 17F, 18C, 19A, 20,22F, 23F and 33F type, or the combination in any of these (according to the typings of Denmark system); Staphylococcus aureus capsular polysaccharide 5,8 and 336 types; The poly-agate D glutamic acid that adds of anthrax bacillus trophophase pod membrane; Mycobacterium tuberculosis fat-arabinose mannan pod membrane; The surperficial oligosaccharide of Plasmodium falciparum (Plasmodium falciparum); A, B, C, X, Y and W135 group meningitis diplococcus capsular polysaccharide or its combination.The compositions of flagellin composition (wherein the flagellin composition comprises at least one cysteine residues and this flagellin composition activation clock sample receptor 5 thus) of flagellin part that is included as at least a portion of flagellin can comprise by with antigen (for example, k antigen, pathogen-related antigen, influenza antigen, banzi virus antigen) side chain carries out chemical modification, but produce thus coupling (also being called chemical conjugation) in this paper as for the present invention be substituted produced on the flagellin (as described above) can compatible reactive group active group, and the antigen that makes.Finish the derivatization (modification) of pod membrane material,, and avoid the most of immunoreactivity structure on the pod membrane material to go to pot so that the fraction of side chain (about 5%) modified.Be contained in compositions of the present invention and be used at least a member who is selected from the cohort of being formed by west nile virus albumen, Lan Jiate (Langat) virus protein, Kun Jin (Kunjin) virus protein, Murray Valley encephalitis virus protein, Japanese encephalitis virus albumen, tick-brone encephalitis virus albumen, dengue fever 1 virus protein, dengue fever 2 virus proteins, dengue fever 3 virus proteins, dengue fever 4 virus proteins, hepatitis C virus albumen and yellow fever virus albumen of can be of method of the present invention (referring to, PCT/US2006/001623 (WO2006/078657) for example).

Flavivirus belongs to flaviviridae, and is made up of about 70 kinds of virus.Mosquito or tick are these viral media of great majority.Several banzi virus are important human pathogen, comprise four kinds of dengue virus (Den1, Den2, Den3 and Den4), yellow heat (YF) virus, Japanese encephalitis (JE) virus, west nile virus (WN, also be called " WNV " in this paper) and tick encephalitis (TBE) virus people such as (, Nat Rev Microbiol 10:789-801 (2004)) Wei Fuer S.C..Flavivirus is divided into many sero-groups based on cross neutralization test, comprises the dengue fever sero-group, and it contains in four kinds of serology and the hereditism goes up different virus, called after Den1, Den2, Den3 and Den4.

Banzi virus be little, have the virus of peplos and icosahedron capsid.The banzi virus genome be sub-thread just-meaning RNA (about 11kb), it is directly translated by the host cell machine after infecting.Viral genome is translated into single polypeptide, and it translates shearing after can being total to-being reached by virus and cellular enzymes, produces three kinds of banzi virus structural protein (housing (C), film (M) and peplos (E) albumen); And seven kinds of unstructuredness albumen (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) people such as (, Annu RevMicrobiol 1990:44-649 (2004)) Wei Fuer.Virocapsid is by the C-protein ingredient, and M-and envelope protein are to be positioned at (people such as Wei Fuer S.C., Nat Rev Microbiol 10:789-801 (2004) on the peplos surface of virion; People such as Kan Bai, Annu Rev.Microbiol.44:649-688 (1990)).The main immunogens of banzi virus is a membrane envelope albumen.

Banzi virus is when its virus envelope protein and receptors bind, and resets pH matter to endosome because of configuration and lower when producing reaction, can enter in the host cell.Configuration changes induces virus and host cell membrane to merge.

The peplos of banzi virus can act as a kind of receptor binding protein, and helps virus and host cell membrane fusion.The envelope protein of banzi virus has common structure (functional domain I, II and III) and the functional character receptors bind and the fusion function of host cell (virus with), and is II class fusion glycoprotein people such as (, Cell 105:137-148 (2001)) Lei Sika.

In the configuration, envelope protein forms on the outer surface of virion with first dimer (Lei Dengren, Nature 375:291-298 before merging; People such as elder brother, Cell 108:717-725 (2002); Dusk, the Ke Pei Chinese mugwort was waited people, Science 302:248 (2003)).Each envelope protein monomer is folded into three structure function territories (functional domain I, II and III), mainly is made up of β-thigh.Functional domain I (in this paper also be called " I " or " DI ") be positioned at this structure central authorities, and in the envelope protein of saccharifying, having N-saccharifying site.The functional domain II of envelope protein (in this paper also be called " II " or " DII ") promote double focusing to turn usefulness into, and have one and merge ring structure, it carries out inserting when the pH-dependency merges in the target host film in virus (people such as Mo Disi, Nature427:313-319 (2004); People such as Bu Ruishali, EMBO J 23:728-738 (2004)).Functional domain III (in this paper also be called " III " or " DIII ") be positioned at the carboxyl terminal of envelope protein.Functional domain III also is called " functional domain B " in early stage antigen mapping research.Functional domain III has several epitopes that can cause virus-neutrality antibody (Raleigh lattice, Adv Virus Res 59:141-175 (2003)).

The functional domain I of tick-brone encephalitis virus envelope protein is corresponding to the amino acid/11-51 of SEQ ID NO:777,, 137-189 and 285-302; The functional domain II of the tick-brone encephalitis virus envelope protein of SEQ ID NO:777 is corresponding to aminoacid 52-136 and 190-284; And functional domain III is corresponding to the aminoacid 303-395 (thunder, people such as F.A., Nature 375:291-298 (1995)) of SEQ ID NO:777.SEQ ID NO:777 is encoded by SEQID NO:778.Step on the amino acid/11-52 of leather 2 flavivirus envelopes proteic functional domain I corresponding to SEQ ID NO:763,, 132-193 and 280-296; Functional domain II is corresponding to aminoacid 53-131 and the 194-279 of SEQ ID NO:763; And functional domain III is corresponding to the aminoacid 297-495 (Mo Disi, people such as Y., Nature 427:313-319 (2004)) of SEQ ID NO:763.Functional domain I, II of other banzi virus (for example west nile virus, Japanese encephalitis, dengue fever 1 virus, dengue fever 3 viruses and dengue fever 4 viruses) and the location of III are based on tick encephalitis envelope protein functional domain and the homology of stepping on leather 2 envelope protein functional domains.Be based on homology when therefore, this paper mentions the proteic functional domain of banzi virus (particularly not being tick-borne encephalitis flavivirus albumen and dengue fever 2 flavivirus envelope albumen) to the functional domain in tick-borne encephalitis flavivirus envelope protein and dengue fever 2 flavivirus envelope albumen.

The functional domain III of the envelope protein of the DEN banzi virus most of banzi virus type-specificity adjacent continuous/advantage neutrality epitope (Luo Lin that encodes, J.T., Adv.Virus Res.59:141 (2003)), comprise four kinds of DEN (DEN1, DEN2, DEN3, DEN4) virus.Flavivirus envelope albumen has high homology.Exemplary envelope protein sequence is SEQ ID NOS:642,763,765,767,769 and 774.

West nile virus (WNV) for sub-thread just-meaning RNA has the virus of peplos.Its prior to nineteen thirty-seven in ugandan Xi Niluo zone, be grown up from the fever women and separate and identify people such as (, Am J Trop Med Hyg 3:9-18 (1954)) Smith's Bernes.

Japanese encephalitis (JE) virus is local in Asia and northern Australia (50,000 cases being arranged approximately, wherein about 10,000 death every year).

Dengue fever (DEN) disease is by banzi virus relevant in four kinds of mosquitos-infection type, the serology, is called DEN-1 (also being called " Den1 " or " Den1 " in this paper), DEN-2 (also being called " Den2 " or " Den2 " in this paper), DEN-3 (also being called " Den3 " or " Den3 " in this paper) and DEN-4 (also being called " Den4 " or " Den4 " in this paper) and causes.Compositions of the present invention, fusion rotein and polypeptide can comprise Den 1 SEQ ID NO:623; Den 1 PR 94 (Puerto Rico, 1994) SEQ ID NO:624; Den 3 SEQ ID NO:626; And Den 4 SEQ ID NO:627.SEQ ID NOS:623,624,625,626 and 627 is the partial function territory III of Den1, Den2, Den3 and Den4 banzi virus.

" EI ", " EII " reach " EIII " and are used for this paper and mean functional domain I, II and the III of West Nile flavivirus envelope protein respectively." JEI ", " JEII " reach " JEIII " and are used for this paper and mean functional domain I, II and the III of japanese encephalitis flavivirus envelope protein respectively." Den1 I ", " Den1 II " reach " Den1 III " and are used for this paper and mean the proteic functional domain I of dengue fever 1 flavivirus envelope, II and III respectively.Equally, name for the functional domain of the envelope protein of other banzi virus, be to quote this banzi virus title to add this functional domain numbering (for example, (tick infection type) TBI (tick infection type), TBII, TBIII, Den2 I, Den2 II, Den2 III) afterwards.

The proteic part of flavivirus envelope can comprise at least a member who is selected from the cohort of being made up of at least a portion of at least a portion of at least a portion of functional domain I, functional domain II and functional domain III.When a certain functional domain during with ＂+＂ name (for example " EIII+ " or " JEIII+ "), the envelope protein that is called " III " partly adds functional domain I and II one or the two the composition of integral body of at least a portion wherein for a kind of this functional domain.For example, " EIII+ " is used for this paper and means at least a portion that compositions of the present invention, fused protein and polypeptide comprise functional domain III and functional domain I respectively." EIII+ " also is called " EI/III "." JEIII+ " also is called " JEI/III ".Equally, when compositions comprised the proteic functional domain of flavivirus envelope, then described functional domain can be the combination of functional domain I, II and III, and can be based on this functional domain name.For example, EI/II comprises the functional domain I and the II of West Nile flavivirus.Be used for functional domain (for example, EIII, JEIII, the Den1 III) Shi Bujia "+" of the envelope protein of compositions of the present invention, fused protein and polypeptide in citation, mean the functional domain that compositions, fused protein and polypeptide comprise that this is quoted from.For example, " Den1 III " means the functional domain III that compositions, fused protein and polypeptide comprise dengue fever 1 virus, but not functional domain I.

The west nile virus envelope protein can comprise, at least aly is selected from the NO:610 by SEQ ID, and it is the EIII+ aminoacid sequence, and italics aminoacid is the functional domain I of envelope protein, and all the other sequences are the functional domain III of envelope protein; SEQ ID NO:611, west nile virus, Stanford, CT also is called " western Buddhist nun sieve S "; SEQ ID NO:612, west nile virus, New York, NY also is called " western Buddhist nun sieve NY "; At least a portion of the member of the cohort of being formed with SEQ ID NO:613, SEQ ID NO:610 is encoded by SEQ ID NO:614.

The indigo plant that is used for the present composition, fused protein and polypeptide adds at least a portion that special virus envelope protein can comprise SEQID NO:615.Elder brother Tianjin virus envelope protein can comprise at least a portion of SEQ ID NO:616.The Murray Valley encephalitis envelope protein can comprise at least a portion of SEQ ID NO:617.Japanese encephalitis's envelope protein can comprise, at least a at least a portion that is selected from the member of the cohort of being made up of SEQ ID NO:618 and SEQ ID NO:619.The tick encephalitis envelope protein can comprise at least a portion of SEQ ID NO:620.The yellow fever virus envelope protein can comprise at least a portion of SEQ ID NO:621.The envelope protein of banzi virus can comprise at least a at least a portion that is selected from the member of the cohort of being made up of SEQ ID NO:622 and SEQ ID NO:643.SEQ ID NOS:615,616,617,618,619,620,621,622 and 643 is the part of the functional domain III of virus envelope protein.

Antigen chemically conjugation to flagellin composition and clock sample receptor stimulating agent is formed.Chemical conjugation (this paper also is called " chemical coupling ") can comprise by reactive group, for example mercapto (as cysteine residues), or the conjugation by elementary (for example aminoterminal) or secondary (for example lysine) group derivatization is carried out.Can use different cross-linking agent with TLR part (for example, TLR agonist) chemically conjugation to protein (for example, antigen, the present composition, HA of the present invention and M2e construct) or other molecule (for example nucleic acid, polysaccharide).Exemplary cross-linking agent is commercially available getting, for example available from Pierre Si (Pierce, Luo Kelan, Ill) person.With antigen chemically conjugation to the method for flagellin composition for knowing, and comprise and use the commercially available cross-linking agent that gets, for example in person described herein.

For example, peptide or proteantigen yoke to flagellin composition of the present invention or clock sample receptor stimulating agent are formed, can see through at least one cysteine residues that flagellin composition or clock sample receptor stimulating agent are formed, with protein (influenza antigens for example, as HA, M2e) at least one cysteine residues, use the technology set up to reach.Can be with protein with first difunctionality sulfydryl-specificity cross-linking agent derivatization; Then with the protein desalination to remove unreacted cross-linking agent; Afterwards this peptide or protein partner are added, and via at least one cysteine residues conjugation.The exemplary reagent that is used for this conjugation method, can be on the market available from Pierre Si (Luo Kelan, Ill), for example BMB (catalog number: 22331), BMDB (catalog number: 22332), BMH (catalog number: 22330), BMOE (catalog number: 22323), BM[PEO] ₃(catalog number: 22336), BM[PEO] ₄22337), DPDPB (catalog number: 21702), DTME (catalog number: 22335), HBVS (catalog number: 22334) (catalog number:.

Perhaps, also can with contain the protein of cysteine and antigen conjugation to be positioned at flagellin composition of the present invention, flagellin, clock sample receptor stimulating agent is formed and clock sample receptor stimulating agent on lysine.The protein that does not contain cysteine residues is with Heterobifunctional amine and sulfydryl-specificity cross-linking agent derivatization.After desalination, will contain partner's adding of cysteine and carry out conjugation.The exemplary reagent that is used for this conjugation method can be available from Pierre Si (Luo Kelan, Ill), for example, AMAS (catalog number: 22295), BMPA (catalog number .22296), BMPS (catalog number: 22298), EMCA (catalog number: 22306), EMCS (catalog number: 22308), GMBS (catalog number: 22309), KMUA (catalog number: 22211), LC-SMCC (catalog number: 22362), LC-SPDP (catalog number: 21651), MBS (catalog number: 22311), SATA (catalog number: 26102), SATP (catalog number: 26100), SBAP (catalog number: 22339), SIA (catalog number: 22349), SIAB (catalog number: 22329), SMCC (catalog number: 22360), SMPB (catalog number: 22416), SMPH (catalog number .22363), SMPT (catalog number: 21558), SPDP (catalog number: 21857), sulfo group-EMCS (catalog number: 22307), sulfo group-GMBS (catalog number: 22324), sulfo group-KMUS (catalog number: 21111), sulfo group-LC-SPDP (catalog number: 21650), sulfo group-MBS (catalog number: 22312), sulfo group-SIAB (catalog number: 22327), sulfo group-SMCC (catalog number: 22322), sulfo group-SMPB (catalog number: 22317), sulfo group-LC-SMPT (catalog number .:21568).

(or in addition alternatively) in addition, peptide or proteantigen also can all exist lysine residue conjugation to the flagellin composition of the present invention or clock sample receptor stimulating agent on this two conjugation partner to form via at least one.With this two conjugation partner and with difunctionality amine-specificity cross-linking agent combination.Then suitable different first conjugate is reached from undesirable agglutinator and be purified into first conjugate.Be used for this conjugation method exemplary reagent can available from Pierre Si (Luo Kelan, I11), for example, 21600), BS BSOCOES (catalog number: ₃(catalog number: 21580), DFDNB (catalog number: 21525), DMA (catalog number: 20663), DMP (catalog number: 21666), DMS (catalog number: 20700), DSG (catalog number: 20593), DSP (catalog number: 22585), DSS (catalog number: 21555), DST (catalog number: 20589), DTBP (catalog number: 20665), DTSSP (catalog number: 21578), EGS (catalog number: 21565), MSA (catalog number: 22605), sulfo group-DST (catalog number: 20591), sulfo group-EGS (catalog number: 21566), THPP (catalog number: 22607).

Similarly, also can there be the carboxyl (for example, glutamic acid, aspartic acid or peptide or proteinic carboxyl terminal) on the partner wherein in peptide or proteantigen and exist amine conjugation to the flagellin composition of the present invention or clock sample receptor stimulating agent on another partner to form via at least one.This two conjugation partner is mixed with suitable Heterobifunctional cross-linking agent.Then suitable different first conjugate is reached from undesirable agglutinator and be purified into first conjugate.Be used for this conjugation method exemplary reagent can available from Pierre Si (Luo Kelan, Ill), for example, 22101), EDC (catalog number: 22980) and TFCS (catalog number: 22299) AEDP (catalog number:.

In addition, carbohydrate antigen also can use technology conjugation to protein of the present invention, flagellin composition, flagellin, the clock sample receptor stimulating agent of quite setting up to form and clock sample receptor stimulating agent via at least one cysteine in the protein.For example, at first with protein to contain at least one sulfydryl-specificity, with the Heterobifunctional cross-linking agent derivatization of at least one diazanyl.Then with polysaccharide or oligosaccharide with oxidizer treatment such as sodium metaperiodate, and produce terminal aldehyde radical.To add to protein then through the carbohydrate of oxidation, and conjugation with this aldehyde take place via being positioned at hydrazine on the cross-linking agent through derivatization.Be used for this conjugation method exemplary reagent can available from Pierre Si (Luo Kelan, I11), for example, 22297), EMCH (catalog number: 22106), KMUH (catalog number: 22111) and PDPH (catalog number: 22301) BMPH (catalog number:.

Other lipopeptid class can be via the amino acid side chain on this peptide chain, and conjugation to proteantigen of the present invention, flagellin composition or clock sample receptor stimulating agent formed or clock sample receptor stimulating agent.Its will use to about the peptide conjugation to described similar strategy of protein and reagent.

TLR can be activated by nucleic acid.For example, TLR3 is activated by bifilar (ds) RNA; TLR7 and TLR8 are activated by sub-thread (ss) RNA; And TLR9 is activated by the CpG DNA sequence.Can use several different technologies, the clock sample receptor stimulating agent of nucleic acid TLR is formed conjugation to proteantigen.For example, for not modified dna molecular, can be with 5 ' phosphate group with water solublity carbonyl diimine EDC (Pierre Si, Rockford, IL, catalog number: 22980) modify, add imidazoles subsequently and form terminal phosphoryl imidazoles.Can terminal amino group be replaced with this reactive group by adding ethylenediamine then.Then can use Heterobifunctional maleimide (cysteine-specificity) and NHS-ester (lysine-specificity) cross-linking agent, will be through the nucleic acid conjugation of the amine-modification cysteine to the protein, as preamble about as described in peptide-protein conjugation.

Perhaps (or extraly), can by in second step with the cystamine substituted ethylene diamine, and sulfydryl is incorporated into 5 ' end.After treating the reduction of disulfide bond, but the sulfydryl conjugation is to the cysteine that is positioned on protein of the present invention, flagellin composition, flagellin, clock sample receptor stimulating agent composition and the clock sample receptor stimulating agent freely, or use Heterobifunctional cross-linking agent conjugation to lysine, as described herein.In addition alternatively (or extraly), can synthesize 5 ' or 3 ' end the synthesis type oligonucleotide of modified base is arranged.These modified bases can comprise can use preceding method and by conjugation to proteinic primary amine or mercapto groups.3 of RNA molecule ' end chemically can be modified, to allow itself and protein or other giant molecule coupling.Be positioned at 3 '-glycol on the ribose residue, can use the sodium metaperiodate oxidation and produce aldehyde radical.Can use the cross-linking agent that contains diazanyl then, but MPBH (Pierre Si of covalent modification carbonyl for example; The Lip river Crane, IL, catalog number: 22305), and then via the free sulfhydryl group of maleimide conjugation to the protein, and with this aldehyde conjugation to protein.Perhaps, can contain the cross-linking agent of isocyanates, for example it also contains for PMPI (Pierre Si of conjugation to proteinic sulfydryl; The Lip river Crane, IL, catalog number: 28100), directly with 3 ' hydroxyl derivatization.

Identified synthesis type micromolecule TLR part (for example, clock sample receptor stimulating agent, clock sample receptor antagonist).For example, imiquimod (imiquimod, it effectively activates TLR7) is with sharp quinoline not special (resiquimod is the activator of TLR7 and TLR8).Synthetic analog with various level of performance and specific these chemical compounds.Micromolecule TLR agonist conjugation to the antigenic ability of institute's interest, be can be depending on the chemical property of this TLR part.Some TLR part can have the active group that can be used in chemical conjugation.For example, not special (gardiquimod) (Yin Weiwojin (InvivoGen) of imiquimod and analog guanidine quinoline thereof; Santiago, CA), with TLR7-reactivity adenine analog CL087 (Yin Weiwojin; Santiago CA) has one-level amino (NH ₂), it can be the target thing that is used for being undertaken by the cross-linking agent that contains imines ester or NHS esters derivatization.At another strategy, the guanidine quinoline is special to be contained the hydroxyl that is exposed out (OH), it can be by containing the cross-linking agent of isocyanates, for example PMPI (Pierre Si; The Lip river Crane, IL, catalog number: 28100) carry out derivatization, with the follow-up protein sulfhydryl that is cross-linked to.

In addition, can arrange the customized of derivant of micromolecule TLR part to synthesize, with crosslinked on the diverse location that novel functional group is attached in this molecule to help.Customized derivant also can comprise can be then used in the group of direct binding to proteantigen, for example maleimide.

The chemical conjugation of antigen and flagellin composition can cause the water solublity of this antigen as the composition of the present composition (for example, being essentially hydrophobic antigen, for example ripe shearing site antigen) to increase.

Comprise its flagellin composition at least a portion of flagellin, wherein this flagellin composition comprises at least one cysteine residues and makes this flagellin composition activation clock sample receptor 5 thus, compositions can comprise a cysteine residues that is arranged in the hypervariable region of flagellin composition.

At least one cysteine residues replaces at least one and has aminoacid in the natural flagellin aminoacid flagellin composition.This cysteine residues can replace at least one and be selected from by the amino acid/11,237,238,239,240 of SEQ ID NO:812,241 and the aminoacid of 495 cohorts of being formed; At least one is selected from by the amino acid/11 of SEQ ID NO:498,240,241,242,243,244 and the aminoacid of 505 cohorts of being formed; At least one is selected from by the amino acid/11 of SEQ ID NO:504,237,238,239,240,241 and the aminoacid of 504 cohorts of being formed; At least one is selected from by the amino acid/11 of SEQ ID NO:815,211,212,213 and the aminoacid of 393 cohorts of being formed; At least one is selected from by the amino acid/11 of SEQ ID NO:820,151,152,153,154 and the aminoacid of 287 cohorts of being formed; At least one is selected from by the amino acid/11 of SEQ ID NO:502,238,239,240,241,242,243 and the aminoacid of 497 cohorts of being formed; At least one is selected from by the amino acid/11 of SEQ ID NO:812,237,238,239,240,241 and the aminoacid of 495 cohorts of being formed.

Flagellin composition or clock sample receptor stimulating agent are formed, and can comprise at least a portion of natural flagellin aminoacid sequence, with this cysteine residues combination.

The present invention wherein this cysteine residues replaces at least one aminoacid in the natural flagellin aminoacid sequence flagellin composition, or wherein this flagellin composition comprises at least a portion of natural flagellin aminoacid sequence and the compositions of this cysteine residues combination, can activate clock sample receptor 5.For example, cysteine residues can be arranged in the D1/D2 functional domain of contiguous amino terminal and carboxyl terminal, away from TLR5 recognizing site (referring to, Figure 75 and 76 for example).In addition alternatively (or extraly), cysteine residues can position hypermutation functional domain tip point (referring to, Figure 75 and 76 for example), be positioned at about aminoacid 273, about 238, about 239, about 240 and about 241 of SEQ ID NO:812.Replace polarity or charge residue, for replace hydrophobic amino acid with cysteine residues for for preferable.In the TLR5 recognizing site, be substituted by least good.

The flagellin (FliC) that derives from salmonella typhimurium STF1 is to be described in SEQ ID NO:812 (accession number: P06179).The TLR5 recognizing site is aminoacid about 79 to 117 and about 408 to about 439.Cysteine residues can replace the aminoacid about 408 to about 439 of SEQ ID NO:812; The aminoacid about 1 and about 495 of SEQ ID NO:812; The aminoacid about 237 to about 241 of SEQ ID NO:812; And/or the aminoacid of SEQID NO:812 about 79 to about 117 and about 408 to about 439; Or with above person combination.

Salmonella typhimurium flagellin STF2 (FljB) is described in SEQ ID NO:498.The TLR5 recognizing site is the aminoacid about 80 to about 118 and about 420 to about 451 of SEQ ID NO:498.Cysteine residues can replace the aminoacid about 1 and about 505 of SEQ ID NO:498; The aminoacid about 240 to about 244 of SEQ ID NO:498; The aminoacid about 79 to about 117 and/or about 419 to about 450 of SEQ ID NO:498; Or with above person combination.

Munich Salmonella flagellin is to be described in SEQ ID NO:504 (accession number: #P06179).The TLR5 recognizing site is the aminoacid of SEQ ID NO:504 about 79 to 117 and about 418 to about 449.Cysteine residues can replace the aminoacid about 1 and about 504 of SEQ ID NO:504; The aminoacid about 237 to about 241 of SEQ ID NO:504; About 79 to about 117; And/or SEQ ID NO:504 about 418 to about 449; Or with above person combination.

Escherichia coli bacterium flagellin is to be described in SEQ ID NO:502 (accession number: P04949).The TLR5 recognizing site is the aminoacid about 79 to about 117 and about 410 to about 441 of SEQ ID NO:502.Cysteine residues can replace the aminoacid about 1 and about 497 of SEQ ID NO:502; The aminoacid about 238 to about 243 of SEQ ID NO:502; About 79 to about 117; And/or SEQ ID NO:502 about 410 to about 441; Or with above person combination.

The bacillus pyocyaneus flagellin is to be described in SEQ ID NO:815.The TLR5 recognizing site is the aminoacid of SEQ IDNO:815 about 79 to 114 and about 308 to about 338.Cysteine residues can replace the aminoacid about 1 and about 393 of SEQ IDNO:815; The aminoacid about 211 to about 213 of SEQ ID NO:815; About 79 to about 114; And/or SEQ ID NO:815 about 308 to about 338; Or with above person combination.

The Listeria monoeytogenes flagellin is to be described in SEQ ID NO:820.The TLR5 recognizing site is the aminoacid of SEQ ID NO:820 about 78 to 116 and about 200 to about 231.Cysteine residues can replace the aminoacid about 1 and about 287 of SEQ ID NO:820; The aminoacid about 151 to about 154 of SEQ ID NO:820; About 78 to about 116; And/or SEQ ID NO:820 about 200 to about 231; Or with above person combination.

Be positioned on the STF2 through testing definite TLR5 recognizing site, describe (referring to, for example, Smith, people such as K.D., natural immunity 4:1247-1253 (2003)) be to be positioned at aminoacid about 79 to about 117, and about 420 to about 451.In addition, Smith, people such as K.D. (natural immunity 4:1247-1253 (2003)) identify the TLR5 recognizing site that is positioned on other flagellin based on sequence homology, and for example STF1 is positioned at aminoacid about 79 to about 117, about 408 to about 439; Bacillus pyocyaneus is positioned at aminoacid about 79 to about 117, about 308 to about 339; L.pneumophila is positioned at aminoacid about 79 to about 117, about 381 to about 419; Escherichia coli are positioned at aminoacid about 79 to about 117, about 477 to about 381; S.marcesens is positioned at aminoacid about 79 to about 117, about 265 to about 296; Bacillus subtilis is positioned at aminoacid about 77 to about 117, about 218 to about 249; And Listeria monoeytogenes is positioned at aminoacid about 77 to about 115, about 200 to about 231.

Determined high the parsing and constructed STF1 (FliC) (SEQ ID NO:812), and can be used as analysis TLR5 by the basis of the position of the identification of flagellin and cysteamine replacement/interpolation.Flagellin similar a kind of " Hili dart " has amino-and c-terminus (referring to, Figure 77 for example) at the end of an arm.The TLR5 recognizing site is the outside that probably is positioned at Hili dart, in towards flagellin amino-and the bending of c-terminus under locate.Hinge region, it is to be positioned at crooked top to TLR5 identification and undesirable.The zone with maximal sequence homology of flagellin is to be arranged in the TLR5 recognizing site.The sequence homology zone of next is to be arranged in D1 and D2 functional domain, and it comprises TLR5 recognizing site and amino-and c-terminus.D1 and D2 functional domain (have or do not contain connexon) are the STF2 Δ. (.SEQ ID NO:500), it can activate TLR5.The zone that least has sequence homology between the flagellin is the hypervariable region.

According to believing, flagellin composition or clock sample receptor stimulating agent are formed the ability that can activate TLR5, can be by making conjugation site (replace at least one amino acid whose cysteine residues in the natural flagellin aminoacid sequence flagellin composition, or the combination of at least a portion of natural flagellin aminoacid sequence and cysteine residues) be kept away from TLR5 or TLR recognizing site and finish.For example, be the STF1 (SEQ ID NO:812) that can get for can highly resolving structure mensuration, this can or reach in hinge region in D1 functional domain, D2 functional domain.In the D1/D2 functional domain, amino-and c-terminus can be away from TLR5 recognizing site (also being called " being positioned at long-range " in this paper), and remove from amino or c-terminus and may make the more close recognizing site in conjugation site, and may disturb the TLR5 activity.In hinge region, the aminoacid about 237 to about 241 of SEQ ID NO:812 is another tips that probably are positioned at " Hili dart ", and about with the distance of TLR5 recognizing site and amino-and c-terminus is identical.This site also may be for keeping the position of TLR5 identification.

For the position in conjugation site, can include aminoacid homogeneity in consideration.As if polarity and charge residue (for example, serine, aspartic acid, lysine) may be exposed to the surface and can be attached antigen.As if hydrophobic amino acid (for example, valine, phenylalanine) may be by embedding, and the interaction of participation structure, and should avoid.

The compositions that the clock sample receptor stimulating agent that comprises the flagellin composition with cysteine residues or have cysteine residues is formed, can activate TLR5 also can be through chemical method and antigen conjugation.

Compositions of the present invention and utilize the method for the present composition can further comprise carrier protein.Carrier protein can be at least a member who is selected from by B subunit, coat protein for mosaic virus of tobacco, rabies virus envelope protein, rabies virus envelope glycoprotein, Elityran, heat shock protein 60, keyhole-limpet hemocyanin and early stage secretion antigen tuberculosis-6 cohorts of being formed of the cross reactivity saltant of tetanus toxoid, vibrio cholera toxoid, diphtheria toxoid, diphtheria toxoid, escherichia coli thermal instability enterogenous endotoxin.

Comprise its compositions (wherein this flagellin composition comprises that at least one cysteine residues makes this flagellin composition activation clock sample receptor 5 thus) for the flagellin composition of at least a portion of flagellin, the lysine that can comprise at least one flagellin composition is replaced by at least a member who is selected from the cohort of being made up of arginine residues, serine residue and histidine residues.

In another embodiment, the present invention is a kind of compositions that comprises it for the clock sample receptor stimulating agent composition of at least a portion of clock sample receptor stimulating agent, wherein this clock sample receptor stimulating agent is formed and is comprised that at least one is arranged in natural clock sample receptor stimulating agent and the cysteine residues of locating of cysteine residues do not occur, and makes this clock sample receptor stimulating agent form activation clock sample receptor thus." composition " is used for this paper and forms at least a portion or the whole clock sample receptor stimulating agent that means clock sample receptor stimulating agent about clock sample receptor stimulating agent.

In one embodiment, (wherein this clock sample receptor stimulating agent composition comprises that at least one is arranged in the cysteine residues of locating that cysteine residues does not appear in natural clock sample receptor stimulating agent in comprising its compositions of forming for the clock sample receptor stimulating agent of at least a portion of clock sample receptor stimulating agent, and make this clock sample receptor stimulating agent form activation clock sample receptor thus) cysteine residues.At least one aminoacid that replaces the natural acid sequence of clock sample receptor stimulating agent composition.This cysteine can replace at least one aminoacid of the clock sample receptor recognizing site of forming away from clock sample receptor stimulating agent.In another embodiment, clock sample receptor stimulating agent is formed and is comprised at least a portion of natural clock sample receptor stimulating agent aminoacid sequence in conjunction with cysteine residues.The clock sample receptor recognizing site that can form away from clock sample receptor stimulating agent with the bonded cysteine residues of natural clock sample receptor stimulating agent.

(wherein this clock sample receptor stimulating agent composition comprises that at least one is arranged in the cysteine residues of locating that cysteine residues does not appear in natural clock sample receptor stimulating agent to comprise its clock sample receptor stimulating agent composition at least a portion of clock sample receptor stimulating agent, and make this clock sample receptor stimulating agent form activation clock sample receptor thus) compositions, (for example further comprise at least a antigen, influenza antigen is as influenza virus conformability memebrane protein, HA, HA1-1, HA1-2, M2, M2e) at least a portion.

In another embodiment, the present invention is a kind of compositions that comprises it for the flagellin composition of at least a portion of flagellin, wherein at least one lysine of this flagellin composition is replaced by at least one arginine, makes flagellin composition activation clock sample receptor 5 thus.

" be substituted " and be used for this paper and form about flagellin, flagellin composition, clock sample receptor stimulating agent or clock sample receptor stimulating agent and mean, at least one aminoacid of flagellin composition (for example lysine) has been modified into another amino acid residue, for example conservative replaces (as arginine, serine, histidine), and forms flagellin composition that is substituted or the clock sample receptor stimulating agent that is substituted composition thus.Can be by producing the recombinant precursor that coding has the flagellin of replacement, by chemical method, produce the protein or the peptide class of at least a portion of flagellin by the protein synthesis technology, or its any combination and make the flagellin composition that is substituted or the clock sample receptor stimulating agent that is substituted is formed.

Lysine residue through a seed amino acid (for example arginine, serine, histidine) replaces can be at least one and is selected from by the lysine 19,41,58,135,160,177,179,203,215,221,228,232,241,251,279,292,308,317,326,338,348,357,362,369,378,384,391 of SEQ ID NO:812 and the lysine of 410 cohorts of being formed.

Flagellin can be its salmonella typhimurium flagellin that comprises SEQ ID NO:816.Lysine residue through a seed amino acid (for example arginine, serine, histidine) replacement, can be at least one is selected from by the lysine 20,42,59,136,161,177,182,189,209,227,234,249,271,281,288,299,319,325,328,337,341,355,357,369,381,390,396,403,414 of SEQ ID NO:816 and the lysine of 422 cohorts of being formed.

Flagellin can be its escherichia coli fliC that comprises SEQ ID NO:814.Flagellin can be its Munich Salmonella flagellin that comprises SEQ ID NO:813.Flagellin can be its bacillus pyocyaneus flagellin that comprises SEQ ID NO:815.Flagellin can be its Listeria monoeytogenes flagellin that comprises SEQ ID NO:820.

The present composition can comprise motif C that it has the motif N of the motif C that is arranged in contiguous flagellin, flagellin, flagellin and motif N the two, the substituted flagellin of lysine in the zone of the functional domain 2 of the functional domain 1 of flagellin, flagellin or its combination in any.The motif C of flagellin and functional domain of motif N and flagellin 1 and functional domain 2 can relate to the activation that TLR5 is subjected to flagellin people such as (, J.Biol.Chem., 279:5667-5675 (2004)) Mu Ersi.

The chemical conjugation of protein, peptide or polypeptide and another molecule can pass through secondary group (for example lysine) derivatization.Some lysine residue is approaching or is arranged in functional domain 1, motif C or motif N (may be important motif with combining of TLR5 at flagellin) in the flagellin.For example, be positioned at the aminoacid 58,135,160 of SEQ IDNO:812 and 410 lysine residue, can replace through at least a member who is selected from the cohort of forming by arginine residues, serine residue, histidine residues.This type of lysine residue of derivatization to flagellin, can lower binding ability or the binding affinity of flagellin and TLR5 with (for example) chemical conjugation antigen, and therefore reduces the inherent immunity reaction by the TLR5 mediation.With another kind of aminoacid (for example arginine, serine, histidine) replace in the flagellin at least one may be near the reciprocal action for mediation and TLR5 of flagellin important zone (for example, motif C, motif N, functional domain 1) lysine residue, can keep or strengthen combining of flagellin and TLR5.In special embodiment, aminoacid replacement replaces for the conservative of carrying out with at least a member who is selected from the cohort of being made up of arginine, serine, histidine.The exemplary commercially available reagent that gets that is used for chemical conjugation is described in this paper.

Some lysine residue is to exist in the functional domain (functional domain 1) in the flagellin, and may be important for the activation of TLR5.For example, be to exist in the functional domain 1 in the position 58,135,160 of SEQ ID NO:812 and 410 lysine residue.This type of lysine residue of derivatization can lower the TLR5 biological activity with (for example) chemical conjugation antigen, and therefore reduces the inherent immunity reaction by the TLR5 mediation.

The lysine residue that can be substituted can comprise the activatory lysine residue of participation TLR5.Be arranged in the lysine residue of motif N (the aminoacid 95-108 of SEQ ID NO:812) and/or motif C (the aminoacid 441-449 of SEQ ID NO:812), can be fit to replace.With some lysine residue in the flagellin (for example, be positioned at the lysine of amino acid position 19,41) replace with (for example) arginine, serine or histidine, can keep combining of flagellin and TLR5, and allow other lysine can be used for and other molecule, for example antigen (as protein) or another kind of molecule (as another kind of protein, peptide or polypeptide) chemical conjugation.

Derive from the segmental X-ray of the F41 crystal structure of the flagellin of salmonella typhimurium, show the functional domain structure (mountain horse Supreme Being, people such as F.A, Nature 410:321 (2001)) of flagellin.The total length flagellin contains 4 functional domains, called after D0, D1, D2 and D3.These functional domains wherein three have in crystal structure and show, make because this structure is an albuminolysis fragment with the total length flagellin.The salmonella typhimurium flagellin is for these regional aminoacid sequences, is shown in down with respect to the number column of SEQ ID NO:812:

D0 contains regional A1 to A55 and S451 to R494

D1 contains regional N56 to Q176 and T402 to R450

D2 contains regional K177 to G189 and A284 to A401

D3 contains regional Y190 to V283

The suitable exemplary lysine that replaces with (for example) arginine, histidine or serine of SEQ ID NO:812 can comprise:

D0 contains 2 lysine residues; K19, K41

D1 contains 4 lysine residues; K58, K135, K160 and K410

D2 contains 14 lysine residues; 177,179,292,308,317,326,338,348,357,362,369,378,384,391

D3 contains 8 lysine residues; 203,215,221,228,232,241,251,279.

The exemplary lysine residue that is suitable for replacing comprises the position 58,135,160 that is positioned at SEQ ID NO:812 and 410 lysine (Jie Qili, people J.Bacteriol.185:4243 (2003) such as S.G.; Tang Naili, people such as M.A., J.Biol.Chem.277:40456 (2002)).Described sequence is to derive from the Swiss-Prot protein knowledge base that network address is http://us.expasy.org/sprot/.Lysine residue that can be modified is with a ^*Indicate.

The exemplary lysine residue that SEQ ID NO:816 is suitable for replacing can comprise:

D0-has two lysines in the position 20,42;

D1-has five lysines in the position 59,136,161,414,422;

D2-have 16 lysines in the position 177,182,189,299,319,325,328,337,341,355,357,369,381,390,396,403 and

Seven lysines of D3-tool are in the position 209,227,234,249,271,281,288.

In another embodiment, the present invention is a kind of compositions that comprises it for the flagellin composition of at least a portion of flagellin, wherein at least one lysine of this flagellin composition is replaced by at least one histidine residues, makes flagellin composition activation clock sample receptor 5 thus.

In another embodiment again, the present invention is the individual interior immunoreactive method of a kind of stimulation, it comprises this individual step is given in a kind of compositions administration of flagellin composition that it is included as at least a portion of flagellin, wherein this flagellin composition comprises at least one cysteine residues, and makes flagellin composition activation clock sample receptor 5 thus.

In another embodiment; the present invention is a kind of method that stimulates individual interior protective immunity; it comprises this individual step is given in a kind of compositions administration of flagellin composition that it is included as at least a portion of flagellin; wherein this flagellin composition comprises at least one cysteine residues, and makes flagellin composition activation clock sample receptor 5 thus.

In another embodiment; the present invention is a kind of method that stimulates individual interior protective immunity; it comprises this individual step is given in a kind of compositions administration of flagellin composition that it is included as at least a portion of flagellin; wherein at least one lysine of this flagellin composition is replaced by at least one arginine, makes flagellin composition activation clock sample receptor 5 thus.

In another embodiment, the present invention is the individual interior immunoreactive method of a kind of stimulation, it comprises this individual step is given in a kind of compositions administration of flagellin composition that it is included as at least a portion of flagellin, wherein at least one lysine of this flagellin composition is replaced by at least one serine residue, makes flagellin composition activation clock sample receptor 5 thus.

In another embodiment; the present invention is a kind of method that stimulates individual interior protective immunity; it comprises this individual step is given in a kind of compositions administration of flagellin composition that it is included as at least a portion of flagellin; wherein at least one lysine of this flagellin composition is replaced by at least one serine residue, makes flagellin composition activation clock sample receptor 5 thus.

In an extra embodiment, the present invention is the individual interior immunoreactive method of a kind of stimulation, it comprises this individual step is given in a kind of compositions administration of flagellin composition that it is included as at least a portion of flagellin, wherein at least one lysine of this flagellin composition is replaced by at least one histidine residues, makes flagellin composition activation clock sample receptor 5 thus.

In other embodiment; the present invention is a kind of method that stimulates individual interior protective immunity; it comprises this individual step is given in a kind of compositions administration of flagellin composition that it is included as at least a portion of flagellin; wherein at least one lysine of this flagellin composition is replaced by at least one histidine residues, makes flagellin composition activation clock sample receptor 5 thus.

Another embodiment of the present invention, be the individual interior immunoreactive method of a kind of stimulation, it comprises the compositions administration that a kind of clock sample receptor stimulating agent that it is included as at least a portion of clock sample receptor stimulating agent is formed and gives this individual step, wherein this clock sample receptor stimulating agent is formed and is comprised at least one cysteine residues, be arranged in natural clock sample receptor stimulating agent and locating of cysteine residues do not occur, and make this clock sample receptor stimulating agent form activation clock sample receptor thus.

Another embodiment of the present invention; be a kind of method that stimulates individual interior protective immunity; it comprises the compositions administration that a kind of clock sample receptor stimulating agent that it is included as at least a portion of clock sample receptor stimulating agent is formed and gives this individual step; wherein this clock sample receptor stimulating agent is formed and is comprised at least one; be arranged in natural clock sample receptor stimulating agent and the cysteine residues of locating of cysteine residues do not occur, and make this clock sample receptor stimulating agent form activation clock sample receptor thus.

In another embodiment, the present invention comprises that with it clock sample receptor stimulating agent (for example for a kind of antigen composition that comprises it at least a portion that comprises the ripe shearing site of hemagglutinin, at least a being selected from by clock sample receptor 1 agonist, clock sample receptor 3 agonist, clock sample receptor 5 agonist, clock sample receptor 6 agonist, the member of the cohort that clock sample receptor 7 agonist and clock sample receptor 9 agonist are formed) compositions that agonist is formed, wherein this clock sample receptor stimulating agent be not clock sample receptor 2 agonists (for example, outer membrane protein complex (OMPC) is as the OMPC of meningococcus).The ripe shearing site of hemagglutinin can comprise at least a member who is selected from the cohort of being made up of the ripe shearing site of influenza A hemagglutinin, the ripe shearing site of influenza B hemagglutinin and the ripe shearing site of influenza C hemagglutinin.

In an embodiment, the antigen of the present composition is formed with agonist and is formed the composition that can be fused protein.In another embodiment, be antigen to be formed chemical conjugation to agonist form.Agonist is formed and is comprised a kind of compositions that comprises it for the flagellin composition of at least a portion of flagellin, and wherein the flagellin composition comprises at least one cysteine residues, and makes flagellin composition activation clock sample receptor 5 thus.Antigen is formed and is further comprised the proteic at least a portion of substrate-2, it can further comprise second agonist composition, it comprises at least a portion (for example, at least a at least a portion that is selected from the member of the cohort of being made up of clock sample receptor 2 agonists, clock sample receptor 3 agonist, clock sample receptor 4 agonist, clock sample receptor 5 agonist, clock sample receptor 6 agonist, clock sample receptor 7 agonist and clock sample receptor 9 agonist) of second kind of clock sample receptor stimulating agent.Substrate-2 albumen can be formed through fusion to this second agonist, or chemical conjugation to the second agonist is formed.

" antigen composition " is used for this paper and means an a certain antigenic part or integral body.

" agonist composition " is used for a part or the integral body that this paper means a certain agonist (for example clock sample receptor stimulating agent).

In an extra embodiment, the present invention is the individual interior immunoreactive method of a kind of stimulation, it comprises forms a kind of it antigen that comprises at least a portion of the ripe shearing site of hemagglutinin that comprises the compositions administration of the agonist composition that comprises clock sample receptor stimulating agent with it and gives this individual step, and wherein this clock sample receptor stimulating agent is not a clock sample receptor 2 agonists.

In other a embodiment; the present invention is a kind of method that stimulates individual interior protective immunity; it comprises forms a kind of it antigen that comprises at least a portion of the ripe shearing site of hemagglutinin that comprises the compositions administration of the agonist composition that comprises clock sample receptor stimulating agent with it and gives this individual step, and wherein this clock sample receptor stimulating agent is not a clock sample receptor 2 agonists.

In another embodiment, the present invention for a kind of it comprise through merge (for example with recombination method) to or chemical conjugation to antigenic at least a portion of HA (for example ripe shearing site) of at least a portion of flagellin, flagellin composition or the hinge region in the flagellin that hinge region has wherein been lacked; And through merging (for example with recombination method) extremely, or chemical conjugation is to flagellin or flagellin composition, for example, and in the amino of flagellin or flagellin composition-or compositions of proteic at least a portion of M2e of carboxyl-end (referring to, Figure 77 for example).

In an extra embodiment, the present invention includes with protein of the present invention, polypeptide and peptide have at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98% and at least about protein, peptide and the polypeptide of 99% sequence homogeneity.

Percentage ratio homogeneity between two aminoacid sequences (or two nucleotide sequences), can measure by sequence is calibrated (for example, the gap can be imported in the sequence of first sequence) according to the right purpose of optimum ratio.Comparison is positioned at the aminoacid sequence or the nucleotide sequence of opposite position then, and the percentage ratio homogeneity between two sequences be by the function of the shared same position number of described sequence (that is, total #x 100 of the #/position of % homogeneity=same position).Can be protein or nucleic acid coding length that the purpose of comparison is calibrated, for reference sequences (for example, the nucleotide sequence of the protein portion of HA (as HA1-1, HA1-2), fused protein, antigen or polypeptide) at least 30% of length, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100%.

The actual specific of two sequences is to can for example using mathematical algorithm to finish by the method for having known.Preferable a, limiting examples of this type of mathematical algorithm is through being described in card Lin Dengren (its entire teachings is to include this paper in way of reference for Proc.Natl.Acad.Sci.USA, 90:5873-5877 (1993)).This algorithm is to incorporate into as in people (its entire teachings is to include this paper in way of reference for nucleic acids research (Nucleic Acids Res.), 29:2994-3005 (2001)) described BLASTN such as Xue's Buddhist and the BLASTX program (2.2 editions).When using BLAST and Gapped blast program, can use individual programs (for example, BLASTN; Can be used in the internet network address of NCBI) parameter preset.In an embodiment, the data base who is searched is irredundant (NR) data base, and the capable setting parameter that is used for sequence alignment is in no filter; Desired value is 10; The literal size is 3; Matrix is BLOSUM62; And a cost (Gap Cost) have the existence value be 11 and the extension value be 1.

The mathematical algorithm that another kind is used for sequence alignment is the algorithm of Myers and Miller (CABIOS (1989), its entire teachings is to include this paper in way of reference).This type of algorithm is to be merged in the ALIGN program (2.0 editions), and this program is the part of GCG (Accelrys, Santiago, California) sequence calibration software bag.When using the ALIGN program to compare aminoacid sequence, be to use PAM120 weighting residue table, the gap length rule of punishing is 12, and the gap rule of punishing is 4.Other algorithm that is used for sequence alignment is known for this skill, and comprises as Tuo Ruilisi and carry (its entire teachings is to include this paper in way of reference for Comput.Appl.Biosci., 10:3-5 (1994)) described ADVANCE and ADAM with spreading out; And through being described in the FASTA of Pearson and Li Puman (its entire teachings is to include this paper in way of reference for Proc.Natl.Acad.Sci USA, 85:2444-2448 (1988)).

Percentage ratio homogeneity between two aminoacid sequences, also can use the GAP program in the GCG software kit (Accelrys, Santiago, California), use Blossom 63 matrixes or PAM250 matrix, and the gap be weighted to 12,10,8,6 or 4 and length be weighted to 2,3 or 4 and finish.In another embodiment, the percentage ratio homogeneity between two nucleotide sequences can be used the GAP program in the GCG software kit (Accelrys, Santiago, California), use the gap be weighted to 50 and length be weighted to 3 and finish.

HA of the present invention encodes, the part of polypeptide or fused protein and the nucleotide sequence of polypeptide of the present invention, can be included under the selective cross condition (for example high harsh property hybridization conditions) and the complement of nucleotide sequence of the present invention or nucleotide sequence of the present invention (SEQ ID NOS:53-58 for example, 64,68,71,72,73,76,78,80,84,87,104,107,110,111,112,115,116,117,119,120,121,122,123,124,125,126,127,128,129,130,131,132,133,137,139,142,143,146,147,150,167,184,185,186,187,188,189,190,191 and 192) and code book invention aminoacid sequence and fused protein (for example, SEQ ID NOS:89-92,95,151-160,177,209, the nucleotide sequence of nucleic acid array hybridizing 210 and 211).Be used for this paper, term " in the hybridization down of low harsh degree ", " in the hybridization down of harsh degree ", " in the hybridization down of the harsh degree of height " or " in the hybridization down of very high harsh degree " be the condition of describing the hybridization and the rinsing that are used for nucleotide sequence.About the guiding of carrying out hybridization (can comprise aqueous and non-aqueous method) can referring to, molecular biology motion now (Current Protocols in Molecular Biology), John Willie father and son, N.Y. (1989), it intactly includes this paper in way of reference.

For the application that needs high selectivity, can use and go up higher harsh degree condition relatively and form crossbred.In being used for some solution, adding organic solvent (for example Methanamide) and can make reaction take place at a lower temperature based on the hybridization of film.High harsh degree condition is that (for example) goes up lower salt and/or higher temperature conditions relatively.High harsh degree be by about 0.02M to about 0.10M NaCl, extremely provide under about 70 ℃ temperature in about 50 ℃.The mismatch limited amount takes place between high harsh degree conditions permit two sequences.For reaching low harsh degree condition, can increase salt density and/or can lower temperature.In harsh degree be to be about 0.1 to 0.25M NaCl in salt density, and about 37 ℃ reached to about 55 ℃ temperature, and low harsh degree is to be about 0.15 to 0.9M NaCl in salt density, and reach to about 55 ℃ temperature temperature range from about 20 ℃.Composition that is used to hybridize and condition, know for practising in this skill personage, and through looking back in people such as Shu Beier difficult to understand (1997, molecular biological brief motion (Short Protocols in MolecularBiology), John Willie father and son, New York N.Y., unit 2.8-2.11,3.18-3.19 and 4-64.9).

" individuality " is used for this paper and can be mammal, for example primates or Nie tooth class (for example rat, mice).In special embodiment, individuality is human.

" effective dose " be meant when individuality is given in its administration when about the amount of the present composition and fused protein, is compositions and the fusion rotein amount or the dosage of the amount that is enough to produce therapeutic efficiency (for example, be enough to stimulate individual in immunoreactive amount).Compositions of the present invention and fusion rotein can single agent or with the multi-agent administration.

Method of the present invention can be by with intestinal or parenteral mode, administration compositions of the present invention and fusion rotein and finish.Especially, route of administration is via oral absorption (for example, beverage, tablet, capsule form) or intramuscular injection said composition and fusion rotein.Other route of administration also is contained in the present invention, comprises intravenous, intradermal, intraarticular, intraperitoneal or subcutaneous route, and nasal administration.Also can use suppository or percutaneous patch.

Compositions of the present invention and fusion rotein, in vitro individual self dendritic cell are given in administration.After dendritic cell are exposed to compositions of the present invention and fusion rotein, described dendritic cell administration can be given this individuality.

Compositions of the present invention and fusion rotein can be individually dosed, maybe can be total to administration and give the patient.Altogether administration is intended to comprise simultaneously or administration in regular turn is indivedual or be the present composition, fusion rotein or the polypeptide of combination.In compositions of the present invention and fusion rotein is under the situation of individually administration, form of medication can be in time close enough is (for example each other, the administration time of compositions and the administration time of fusion rotein are approaching) so that immunoreactive effect is maximum in the individuality for stimulating.Also be susceptible to multiple route of administration (for example, intramuscular, oral, intradermal) and can be used for administration compositions of the present invention and fusion rotein.

Compositions of the present invention and fusion rotein can be separately or are admixture with known excipient (for example be suitable for pharmaceutically (or on physiology) acceptable organic or inorganic supporting agent material that intestinal or parenteral are used, it can not react nocuously with extract).Suitable pharmaceutically acceptable supporting agent comprises water, saline solution (such as Ringer's mixture), alcohols, oils, gelatin and carbohydrate such as lactose, amylose or starch, fatty acid ester, hydroxy methocel and polyvinyl pyrrolidone.This type of preparation can be through sterilization, and (if wish) with can not mix such as lubricant, antiseptic, tranquilizer, wetting agent, emulsifying agent, the salt that is used to influence osmotic pressure, buffer, coloring agent and/or aroma substance etc. with the adjuvant that compositions of the present invention, fusion rotein or polypeptide react nocuously.If wish, also can be with preparation and other active substance combination in order to the minimizing metabolic degradation.Compositions of the present invention and fusion rotein can be by oral administrations (for example being a kind of beverage), intramuscular or peritoneal injection, or carry out administration through intranasal delivery side formula.Compositions and fusion rotein are independent, or when making up with admixture, can single agent or more than potion administration a period of time, (for example to give desirable effect, alleviate prophylaxis of viral infections, alleviate for example symptom of influenza or flaviviridae infections of viral infection).

When needs or when wishing that parenteral is used, the admixture that is specially adapted to compositions and fusion rotein is injectable, sterile solution (preferably being oiliness or aqueous solution), and suspension, emulsion or implant (comprising suppository).Especially, the supporting agent that is used for the parenteral administration comprises, dextrose aqueous solution, saline solution, pure water, ethanol, glycerol, propylene glycol, Oleum Arachidis hypogaeae semen, Oleum sesami, polyoxyethylene blocks polymer etc.Ampoule is unit dose easily.Also compositions, fusion rotein or polypeptide can be incorporated in the liposome, or via percutaneous group Pu or patch administration.Be applicable to that medical admixture of the present invention is known in this skill personage by habit, and through being described in (for example) pharmacy science (Pharmaceutical Sciences) (the 17th edition, mark publishing company (Mack Pub.Co.), Easton, PA) and WO 96/05309, its teaching is to include this paper in way of reference.

Compositions of the present invention and fusion rotein can be in giving individual immune system in order to the present composition, fusion rotein and polypeptide are represented, and in individual on the immunoreactive support body of generation administration give individuality.Representing of the present composition, fusion rotein and polypeptide will preferably comprise the antigenic portions that exposes virus protein, to produce antibody.The composition of the present composition, fusion rotein and polypeptide (for example, PAMP and virus protein), the physical location on support body is close to each other.Compositions of the present invention and fusion rotein by covalently or non-covalently property connection, and can be affixed on this support body.Preferably, this support body is a biological compatibility." biological compatibility " is used for this paper and means this support body and do not produce immunoreation (for example, make antibody) in individuality.Support body can be Biodegradable substrate supporting agent, for example polymer beads or liposome.Support body can further comprise Alumen or suitable adjuvant.Support body can be virus (for example adenovirus, poxvirus, Ah cutting down's virus), antibacterial (for example Salmonella) or nucleic acid (for example plasmid DNA).

Individual dosage and frequency (single agent or multi-agent) are given in administration, and visual various different factors comprise before being exposed to a certain antigen, virus protein the duration of viral infection, previous treatment of viral infections, the route of administration of compositions, fusion rotein or polypeptide; Individual size, age, sex, health, body weight, body-mass index and diet; Virus exposure, viral infection and the specific virus that causes infection are (for example, banzi virus, influenza virus) characteristic and symptom degree, or the treatment or the infection of another antigen (for example influenza antigens), the kind for the treatment of simultaneously, due to illness poison exposes the complication that causes, viral infection or appear, or other health-relevant problem and changing to some extent.Other treatment regime or agent can be used for combining with method and composition of the present invention, fusion rotein or polypeptide.For example the administration of compositions and fusion rotein can be followed the use of other viral therapy agent or agent, to treat and to be exposed to this antigen, for example flaviviridae infections is relevant or because of its disease that causes (for example, hyperpyrexia, paralysis, DHF, meningoencephalitis) or influenza infection.Having determined the adjustment and the operation of dosage (for example frequency and time length), is to belong to practising in this skill personage's capabilities.

Influenza virus is the single-stranded RNA virus that belongs to Orthomyxoviridae family.Influenza virus is divided into three types (A, B, C), depends on its ribonucleoprotein in virus (RNP) and the antigenic antigenic specificity of substrate (M).Influenza A virus can infect human and several other mammals (comprising pig and horse) in nature, and many various avian species, and causes infectious disease and epidemic diseases in human group.As if influenza B virus only infects human in nature, and can cause human infectious disease.Influenza C virus is separated with pig from the mankind, but generally infectious disease can not take place, and causes gentle disease usually the mankind.

Sophisticated influenza virus particles has peplos to coat, and has multi-structure, particle size range from 80 to 120nm.Single-stranded RNA genome and spiral nucleoprotein closely associate, and exist with ribonucleoprotein (RNP) sections that seven (influenza C) or eight (influenza A and B) separate, and each must exist successfully to carry out virus replication.The genome of merogenesis is encapsulated in the outer lipoprotein envelope.The inboard of lipoprotein envelope outside stromatin 1 (MP1 or also be called " M1 " in this paper) is arranged in, and combine with RNP.

Hemagglutinin (HA) is a kind of surface glycoprotein that is positioned on the virus (for example, influenza virus), and it is responsible for being bonded to the refreshing arginine (NeuNAc of N-acetyl group on the host cell; Also be called " sialic acid " in this paper) with the follow-up virus and the fusion of host's film.HA can cause erythrocyte caking or agglutinative usefulness and obtain its name with it.Influenza HA is the trimer of being made up of three monomers (HA0) subunit.HA carries out two kinds of important function in course of infection: with cell membrane sialyloligosaccharide receptors bind, and the fusion of virus and host's film.With after host cell membrane combines, host cell membrane is involved in virus in the endosome at the HA trimer, and attempt digests wherein content by the inside acidify with endosome, and it is transported to lysosome in the host cell.Yet, lysosomal sour environment makes the HA unease, cause HA0 partly to stretch and expose protease-responsive site (ripe shearing site), it can be sheared and form by banded HA1 of single disulfide bond and HA2 subunit (Willie by the host protein enzyme, D.C. wait the people, Annu.Rev.Biochem.56:365-394 (1987)).Shearing betides particular amino acid residue, and produces hydrophobicity amino terminal (for the HA2 subunit).The hydrophobicity end of this HA2 mediates the intermembranous fusion of endosome of peplos and host cell, and the content of virion is released in the Cytoplasm, and this process is called sloughs shell.Therefore, HA to be cut into infectious institute essential.

Determined several viral hemagglutination elements crystal structure (referring to, Wilson for example, people such as I.A., nature (Nature) 289:366-373 (1981); Old, people such as J., cell 95:409-417 (1998); Breathe out people such as Y., The EMBO Journal 21:865-875 (2002); The Ruse that, R.J. virusology 325:287-296 (2004); And Ke Kesi, N.J. wait the people, in: microbiology and the infected by microbes (Toply and Wilson ' s Microbiology and Microbial Infections) with Wilson founds in Top, people such as B.W.J Ma Di are compiling, volume 1 (the 9th edition) New York, NY, the Oxford University publishes, the 32nd chapter, p.634 (1998)).X-radiocrystallography structure shows, HA is folded into that two structures are formed or functional domain-ball head and fibrous handle (referring to, Fig. 1 for example).Ball head comprises HA1, and it comprises HA1 and the bonded part of sialic acid (also being called " receptor binding site or functional domain " or " sialic acid binding site or functional domain ") and antiparallel β-flap.Fibrous handle is near viromembrane, and is made up of the part of whole HA2 and HA1, comprises the shearing site between HA1 and HA2.

The known hypotype (H1-H15) that 15 kinds of influenza A HA are arranged, they total about 40 are to about 60% sequence homogeneity (BULL. World Health Organization (WHO) of World Health Organization (WHO) (World Health OrganizationBULL.World Health Organ.), 58:585-591 (1980)).Isolate the influenza virus that contains whole 15HA hypotypes from birds (H5, H7 and H9), horse (H3 and H7), sea dog (H3, H4 and H7), whale (H1 and H13) and pig (H1, H3 and H9).The hypotype of influenza A virus generally is specific antigen epitope (H, 15 main types) and neuraminidase (N, the about 9 main types) name according to HA.For example, hypotype comprises influenza A (H2N1), A (H3N2), A (H5N1), A (H7N2), A (H9N2), A (H1/H0), A (H3/H0) and A (H5/H0).In nearest a century, have three kinds of hypotypes to cause popular: H1 is in 1918 and 1977; H2 in 1957 and H3 in 1968.In 1997, H5 birds virus was reached in 1999, and H9 virus causes area, Hong Kong outburst respiratory tract disease.The HA that is derived from the Type B influenza virus isolates from the mankind and sea dog, and it is not distinguished into hypotype.

May start a kind of antibody response of the HA of antagonism ball head through the host of influenza infection, it is by the interaction between blocking-up HA and host cell, that is the infectivity of this virus that neutralizes, and protects this host follow-up no longer by same strain viral infection.Because the correctness that Influenza Virus RNA is duplicated is low and speed is high, virus is carried out the less important sudden change of HA gene consistently, and it still keeps ball head structure and host cell effect, monitors but may make progeny virus escape immune system.That these rite-directed mutagenesises claim is " antigenic drift (antigenic drift) ".In addition, if single host is infected by the influenza A virus of two different strains simultaneously, then may be because exchange RNA sections (or gene) between the influenza A virus of reprovision or different strains, and cause germinating new virus subtype.Because of the virus that reprovision germinates the neoantigen sex experience is presented to the human immunity system, it can cause high morbidity and mortality rate usually.The antigenic change of this type fierceness is called " antigenicity transfer (antigenic shift) ".Because the Type B influenza virus is almost exclusively only in mankind circulation, so wait virus can't carry out reprovision with the animal strain, and so change by antigenic drift.

Immunity to HA can reduce possibility of infection, and if the disease seriousness when infection takes place really.HA is important antigenicity target, and the effectiveness of vaccine depends on the antigenicity pairing of vaccine strain and circulation strain.Because the hemagglutination fibroin carries out the immune defence that antigenicity is transferred and drift motion is hidden the host easily, so traditional vaccine must be basis and annual the renewal with the strains of influenza viruses that is circulating.The annual influenza virus vaccine that upgrades not only costs dearly, and they also need a large amount of manufacturing times and make capital construction.Based on the vaccine combination in the unmanifest district of virus, can provide the protective effect of wide scope cross reactivity.

Opposite with the HA ball head, near the change of the amino acid residue the ripe shearing site of HA can be restricted because of the function constraint.Near the ripe shearing site of HA amino acid residue influence identification, and the therefore property sheared sheared by the host protein enzyme of this site.This protease because virus is not encoded is so near the amino acid residue that changes the ripe shearing site is to be restricted.As a result, one section about 20 amino acid whose peptide of crossing over ripe shearing site still keeps stable (WO2004/080403 on gene between the influenza virus of identical HA hypotype; Strange people, the J Virol 79:7380-7388 (2005) of waiting of class) or be peptide class people such as (, Immunol Letters 60:127-136 (1998), people such as that Ji, Scand J Immunol40:281-291 (1994)) lotus gas of difference.

The second highly intrinsic antigen of A type influenza virus is the outer functional domains (M2e) of substrate 2 proteic born of the same parents.M2 is with the 97-aminoacid protein of low expression level in ripe virion, and level is much higher in infected cells.M2 albumen forms with first tetramer, and its function is a kind of for the ion channel of virus replication for necessity, and therefore, sudden change among the M2e and the sudden change toleration that is not so good as among the HA are equally high.The outer functional domain (M2e) of 24-extracellular domain amino acid has high conservative at most A type strains of influenza viruses.In mammal, the M2e of native form is low immunogenicity.In the animal model of A type influenza infection, can give passive protection (Cui Nuo, people such as J.J., J Virol 64:1375 (1990) at the antibody of M2e; Liu, people such as W.P., Immunol Lett 93:131 (2004)), be not by virus and the protecting from infection property of neutralizing, but by killing infected cell and destroying life cycle (Qi Beidi, people such as S.L., the J.Virol.62:2762 (1998) of virus; Jie Gele, people such as A.N., J Immunol 172:5598 (2004)), it can reach (Jie Gele, people such as A.N., J Immunol 172:5598 (2004)) by antibody-dependency NK cytoactive.Comprise that the proteic compositions of M2e can limit influenza A severity of disease, host immune response can be developed adaptation immunity for advantage neutrality influenza antigens (HA).

The infection that causes because of influenza infection in order to management and control and the strategy of disease there is no remarkable change in 40 years in the past.Because the Seasonal Characteristics of influenza disease, dissimilar (A and the B) that threaten the influenza virus of human group, and the genic instability of various virus, so the strain that must will may circulate in human group in season in influenza on the horizon every year based on epidemiology prediction is modulated polyvalent vaccine again.Vaccine is from selected prototype virus strain reserve, grows in to contain in embryo's egg and make.Therefore, present strategy has several restrictions, comprising: (a) be dependent on the uncertain prediction for the strain that will circulate; (b) rely on and to make suitable Strain grow in ability in the egg; (c) production system based on egg has the danger that pollution takes place product; (d) product of producing in egg can not be used for egg individuality hypersensitive; And (e) typical polyvalent vaccine can not give to resist human group it be there is no the obvious danger of protection of the viral communication strain of the immunity that is pre-existing in.

The main protectiveness of present available influenza vaccines consists of viral hemagglutination element (HA).Effectively vaccine may not only comprise strain specificity HA, also comprises intersecting protective antigen, for example M2e and ripe shearing site.At present more believable based on the method for chicken, tool economic benefit and the vaccine manufacture method that can enlarge scale also are preferable.Compositions of the present invention, fusion rotein and polypeptide provide the compositions that comprises HA, M2e and the proteic ripe shearing site of influenza, but its immune response stimulating is especially, individual interior at the antigenic protective immunological reaction of a plurality of influenzas.

The teaching of all patent cases that this paper quoted from, open application case and list of references is all intactly included this paper in way of reference.

Embodiment

Embodiment 1: the part of the natural hemagglutinin of design A type influenza virus

Materials and methods

Design is for HA1-1,1-2 and the 1-3 ball head construct of A/ Puerto Rico/8/34 (H1N1)Strains of influenza viruses A/ Puerto Rico/8/34 (PR8) is the mice-adapted strain of A type influenza virus ten minutes characterization.Use ripe PR8HA (SEQ ID NO:1) (people such as Gan Bailin, 2005 science 303:1838-1842; PDB accession number 1RU7) definite crystal structure, binding molecule modelling data base determines the border of three PR8 ball head constructs.The complete list that solves crystal structure of A type influenza virus haemagglutinin molecule, can be referring to Protein Data Bank (Protein Data Bank, PDB) or international biotechnology information centre (National Center for Biotechnology Information, NCBI) structure website (http://www.ncbi.nlm.nih.gov/ structure).

The three-dimensional crystalline structure of PR8 HA is to use the Cn3D program to watch in the NCBI website.Trimer HA molecule has mushroom shaped.Ball head is meant and is positioned at hemagglutinin molecule top, be assembled into the part of mushroom head, and coiled coil type shank is meant, this group of molecules is dressed up the bottom (Fig. 1) of mushroom handle.Ball head contains and the bonded substrate binding site of the sialic acid of cell surface, is important and therefore enter for virus.In addition, most of neutrality antibody epitopes be positioned near and surround this substrate binding site, make ball head become the good target thing of protectiveness vaccine.Ball head contains and most of HA1 peptide, and it comprises the residue of (for example) label in PR8 from E51 to K327.

The monomer structure (it includes ball head) of PR8 chain A is to be used for guiding design PR8HA1-1,1-2 and 1-3 construct (Fig. 1).Described HA construct is through being designed to have the functional domain border in ball head, so that when this molecule was expressed in host cell, coded albumen mass-energy was in spontaneous folding or stretching, extension in vitro, to simulate natural configuration.Structure function territory (also being called " functional domain " in this paper) in the protein is, tool oneself stability in the overall structure, and often be independent of the assembly that these protein chain other parts fold.Most of functional domains can be classified into folding.Many functional domains are not peculiar by the protein institute of term single gene or the manufacturing of term single gene family, but appear in the range protein.Functional domain is often because they play an important role in the proteinic biological function under it, and named or independent; For example, the substrate combined function territory of hemagglutinin can participate in and the combining of substrate.Because functional domain is can the oneself stable,, or by genetic engineering and the association of other carrier protein, and produce mosaic type protein between a certain protein and another protein so functional domain can exchange.Functional domain can be made up of nothing, one section perhaps many structural motif.In the case of influenza virus haemagglutinin construct design, be to keep many structural motifs.It is to instruct by known crystal structure that the border is selected.In the case of PR8, be folded into from the residue (SEQ ID NO:1) of E51 to K327, a kind of tight structure that can distinguish with HA molecule other parts, and therefore for being used to design the focus area of HA construct.

Design is for HA1-1,1-2 and the 1-3 ball head construct of A/ Vietnam/1230/2004 (H5N1)Being used for designing Vietnam/1230/2004 (H5N1) ball head construct HA structure as a reference, is through being described in people such as Shi Tifensi, 2006, and science 312:404-410 (MMDB numbering 38730; PDB accession number 2FK0).About as described in the PR8 ball head construct, use the openly crystal structure of ripe A/VietNam/1203/2004HA (SEQ ID NO:2) as preamble, binding molecule modelling data base determines the border of three Vietnam's ball head constructs.To be used to keep the secondary and the tertiary structure identical construction standard in this structure function territory, be applied to design described Vietnam construct.Though different hemagglutinin molecules, its residue character or number that comprises hemagglutinin may be inequality; Unitary construction is quite similar.Therefore, the border need be positioned on the structure with the HA molecule quite, but numeral go up different positions on the functional domain border of design Vietnam ball head construct.

Design makes up for HA1-1,1-2 and the 1-3 ball head of A/ Indonesia/5/2005 (H5N1) BodyThe crystal structure of Indonesia HA is not solved as yet.Generally, when the crystal structure of given HA molecule when not solved as yet, can use to have the obtained structure of high homogeneity and guide the construct design.In the case of design Indonesia ball head construct, the immediate structure that obtains is A/ Vietnam/1203/2004 (people such as Shi Tifensi, 2006. science 312:404-410; MMDB numbering 38730; PDB accession number 2FK0), itself and A/ Indonesia/5/2005 are same hypotype.

For designing the ball head construct of this HA, at first with primary sequence and the A/ Vietnam/1203/2004HA (H5VN of Indonesia HA (SEQ ID NO:3); SEQ ID NO:2) molecule is calibrated (primary sequence homogeneity: 96.13%).A/ Vietnam/1203/2004HA (H5VN; SEQ ID NO:2) with A/ Indonesia/5/2005 (H5IN; SEQ ID NO:3) calibration of primary sequence is to use CLUSTALW to carry out and is shown in down, (*) asterisk=homogeneity wherein, (:) colon=conservative replacement; (.) fullstop=weak conservative replaces; And (space)=divergence replaces.

50

H5IN MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILE

H5VN MEKIVLLFAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILE

*******:******************************************

100

H5IN KTHNGKLCDLDGVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKAN

H5VN KKHNGKLCDLDGVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKAN

*.*************************************************

150

H5IN PTNDLCYPGSFNDYEELKHLLSRINHFEKIQIIPKSSWSDHEASSGVSSA

H5VN PVVNDLCYPGDFNDYEELKHLLSRINHFEKIQIIPKSSWSSHEASLGVSSA

*.*******.******************************.**** *****

200

H5IN CPYLGSPSFFRNVVWLIKKNSTYPTIKKSYNNTNQEDLLVLWGIHHPNDA

H5VN CPYQGKSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDA

**** *..********************:**********************

250

H5IN AEQTRLYQNPTTYISIGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILK

H5VN AEQTKLYQNPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILK

****:**********:***********:**********************

300

H5IN PNDAINFESNGNFIAPEYAYKIVKKGDSAIMKSELEYGNCNTKCQTPMGA

H5VN PNDAINFESNGNFIAPEYAYKIVKKGDSTIMKSELEYGNCNTKCQTPMGA

****************************:*********************

350

H5IN INSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQRESRRKKRGLFG

H5VN INSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQRERRRKKRGLFG

**************************************** *********

400

H5IN AIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNS

H5VN AIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNS

**************************************************

450

H5IN IIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMEN

H5VN IIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMEN

**************************************************

500

H5IN ERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESIRN

H5VN ERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESVRN

***********************************************:**

550

H5IN GTYNYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMMA

H5VN GTYDYPQYSEEARLKREEISGVKLESIGIYQILSIYSTVASSLALAIMVA

***:********************************************:*

568

H5IN GLSLWMCSNGSLQCRICI(SEQ ID NO：3)

H5VN GLSLWMCSNGSLQCR---(SEQ ID NO：2)

***************

Design is for HA1-1,1-2 and the 1-3 ball head of A/ New Caledonia/20/1999 (H1N1) ConstructThe crystal structure of New Caledonia HA is not solved as yet.For designing the ball head construct of this HA, at first with New Caledonia HA (H1NC; SEQ ID NO:4) primary sequence, the closely related HA molecule that has been solved with two kinds of its structures of identical hypotype (primary sequence homology〉85%), particularly H1N1 1918 viruses (come that/1/18 identical) (SEQ ID NO:5) and A/ Puerto Rico/8/34 virus (H1PR8 with A/ south Caro; SEQ ID NO:1) (people such as Gan Bailin, science 303,1838-42 (2004)) calibrates.A/ south Caro comes that/1/18:MMDB numbering 26943, PDB accession number: 1RUZ; And PR8, MMDB numbering: 26941 with PDB accession number: 1RU7.Primary sequence calibration is to use CLUSTALW to carry out and is shown in down, (*) asterisk=homogeneity wherein, (:) colon=conservative replacement; (.) fullstop=weak conservative replaces; And (space)=divergence replaces.

50

H1N CMKAKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLL

H1PR8 MKANLLVLLSALAAADADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLL

***:*****.:::*: **********************************

100

H1NC EDSHNGKLCLLKGIAPLQLGNCSVAGWILGNPECELLISKESWSYIVETP

H1PR8 EDSHNGKLCRLKGIAPLQLGKCNIAGWLLGNPECDPLLPVRSWSYIVETP

********* **********:*.:***:******:*:. .*********

150

H1NC NPENGTCYPGYFADYEELREQLSSVSSFERFEIFPKESSWPNHTVTGVSA

H1PR8 NSENGICYPGDFIDYEELREQLSSVSSFERFEIFPKESSWPNHNTNGVTA

　 *.*** **** * ******************************...**:*

200

H1NC SCSHNGKSSFYRNLLWLTGKNGLYPNLSKSYVNNKEKEVLVLWGVHHPPN

H1PR8 ACSHEGKSSFYRNLLWLTEKEGSYPKLKNSYVNKKGKEVLVLWGIHHPPN

:***:************* *:* **:*.:****:* ********:*****

250

H1NC IGNQRALYHTENAYVSVVSSHYSRRFTPEIAKRPKVRDQEGRINYYWTLL

H1PR8 SKEQQNIYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQAGRMNYYWTLL

:*: :*:.********:*:*.********:******* **:*******

300

H1NC EPGDTIIFEANGNLIAPWYAFALSRGFGSGIITSNAPMDECDAKCQTPQG

H1PR8 KPGDTIIFEANGNLIAPMYAFALSRGFGSGIITSNASMHECNTKCQTPLG

:**************** ******************.*.**::***** *

350

H1NC AINSSLPFQNVHPVTIGECPKYVRSAKLRMVTGLRNIPSIQSRGLFGAIA

H1PR8 AINSSLPYQNIHPVTIGECPKYVRSAKLRMVTGLRNTPSIQSRGLFGAIA

*******:**:************************* *************

400

H1NC GFIEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIE

H1PR8 GFIEGGWTGMIDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNTVIE

**********:***********************************:***

450

H1NC KMNTQFTAVGKEFNKLERRMENLNKKVDDGFLDIWTYNAELLVLLENERT

H1PR8 KMNIQFTAVGKEFNKLEKRMENLNKKVDDGFLDIWTYNAELLVLLENERT

*** *************:********************************

500

H1NC LDFHDSNVKNLYEKVKSQLKNNAKEIGNGC--------------------

H1PR8 LDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCDNECMESVRNGTY

******************************

550

H1NC --------------------------------------------------

H1PR8 DYPKYSEESKLNREKVDGVKLESMGIYQILAIYSTVASSLVLLVSLGAIS

565

H1NC ---------------(SEQ ID NO：4)

H1PR8 FWMCSNGSLQCRICI(SEQ ID NO：1)

Design makes up for HA1-1,1-2 and the 1-3 ball head of A/ winconsin/67/2005 (H3N2) BodyThe crystal structure of winconsin HA is not solved as yet.For designing the ball head construct of this HA, at first with winconsin HA (H3Wis; SEQ ID NO:6) primary sequence, and the closely related HA molecule that has been solved with a kind of its structure of identical hypotype (primary sequence homogeneity: 81.16%), A/X31 hypotype H3N2 (H3X31 particularly; SEQ ID NO:7) (PDB accession number: 1VIU) calibrate.Winconsin HA sequence is to use CLUSTAL W and A/X31 HA calibration and is shown in down, (*) asterisk=homogeneity wherein, (:) colon=conservative replacement; (.) fullstop=weak conservative replaces; And (space)=divergence replaces.Each aminoacid among the X31 is through finding to have corresponding pairing in A/ winconsin/67/2005 sequence.Then use the functional domain border of X31 structure to authenticate functional domain border at A/Wisconsin/67/2005.

50

H3Wis QKLPGNDNSTATLCLGHHAVPNGTIVKTITNDQIEVTNATELVQSSSTGG

H3X31 QDLPGNDNSTATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSSSTGK

*.**********************:*****:******************

100

H3Wis ICDSPHQILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPY

H3X31 ICNNPHRILDGIDCTLIDALLGDPHCDVFQNETWDLFVERSKAFSNCYPY

**:.**:**** :***********:** ***:.**********:******

150

H3Wis DVPDYASLRSLVASSGTLEFNDESFNWTGVTQNGTSSACKRRSNNSFFSR

H3X31 DVPDYASLRSLVASSGTLEFITEGFTWTGVIQNGGSNACKRGPGSGFFSR

******************** *.*.**** *** *.**** ....****

200

H3Wis LNWLTHLKFKYPALNVTMPNNEKFDKLYIWGVHHPGTDNDQIFLHAQASG

H3X31 LNWLTKSGSTYPVLNVTMPNNDNFDKLYIWGIHHPSTNQEQTSLYVQASG

*****: .**.********::********:***.*:::* *:.****

250

H3Wis RITVSTKRSQQTVIPNIGSRPRIRNIPSRISIYWTIVKPGDILLINSTGN

H3X31 RVTVSTRRSQQTIIPNIGSRPWVRGLSSRISIYWTIVKPGDVLVINSNGN

*:****:*****:******** :*:.**************:*:***.**

　　　　　　　　　　　　　　 300

H3Wis LIAPRGYFKIRSGKSSIMRSDAPIGKCNSECITPNGSIPNDKPFQNVNRI

H3X31 LIAPRGYFKMRTGKSSIMRSDAPIDTCISECITPNGSIPNDKPFQNVNKI

*********:*:************..* ********************:*

329

H3Wis TYGACPRYVKQNTLKLATGMRNVPEKQTR(SEQ ID NO：6)

H3X31 TYGACPKYVKQNTLKLATGMRNVPEKQT-(SEQ ID NO：7)

******:*********************

The result

The description on the functional domain border of PR8 HA constructSelected boundary function territory be in following sequence (SEQ ID NO:1), emphasize as follows: HA1-1The border is to add single underscore (S53-R324 of SEQ ID NO:1);

The border is to add double underline (K62-S284 of SEQ ID NO:1); HA1-3The border is to add thick underline (N101-G276 of SEQ ID NO:1).The detailed description in each subunit design and boundary function territory sees also hereinafter.

60

MKANLLVLLSALAAADADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLED SHNGKLCR

120

L

QLGKCNIAGWLLGNPECDPLLPVRSWSYIVETP NSENGICYPGDFIDYEELRE

180

QLSSVSSFERFEIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTEKEGSYPKLKNS

240

YVNKKGKEVLVLWGIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQA

300

GRMNYYWTLLKPGDTIIFEANGNLIAPMYA FALSRGFG

NASMHECNTKCQTPLG

360

AINSSLPYQNIHPVTIGE CPKYVRSAKLRMVTGLRNIPSIQSRGLFGAIAGFIEGGWTGM

420

IDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNTVIEKMNIQFTAVGKEFNKLEKRM

480

ENLNKKVDDGFLDIWTYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGC

540

FEFYHKCDNECMESVRNGTYDYPKYSEESKLNREKVDGVKLESMGIYQILAIYSTVASSL

565

VLLVSLGAISFWMCSNGSLQCRICI(SEQ ID NO：1)

PR/8HA1-1 construct (SEQ ID NO:8)For this construct, be by before the cysteine of the position 59 of SEQ ID NO:1, making the aminoterminal truncate, and behind the cysteine of the position 319 of SEQ ID NO:1, make the c-terminus truncate, and keep four (4) individual intrinsic disulfide bond.The disulfide bond that is kept lists as follows: the C59-C291 of SEQ ID NO:1; C72-C84; C107-C152 and C295-C319.Use aforementioned crystal structure of quoting and molecular model to make data base (MMDB) and identify three grades and secondary structure, the amino terminal truncate is the serine that is positioned at the position 53 of SEQ ID NO:1, and it is close to by the formed curling corner of the residue 50-52 of SEQ IDNO:1 (LED).The complete cancellation of this truncate is by the residue 44-49[THSVNL (SEQ ID NO:214) of SEQ ID NO:1] the β-thigh before being next to of forming.This strand crossed over the residue 53-58[SHNGKL (SEQ ID NO:215) of SEQ ID NO:1], and the residue 59-62[CRLK of reservation and SEQ IDNO:1 (SEQ ID NO:216)] associating complete β-thigh.Though the residue 53-58[SHNGKL (SEQ ID NO:215) of SEQ ID NO:1 in structure] be defined as random coil, but these residues are simulated β-thighs and are therefore supplied further stable residue 307-310[PYQN by SEQ ID NO:1, SEQ ID NO:217], and the residue 320-323[PKYV of SEQ ID NO:1, SEQ ID NO:218] β-flap of being defined.The carboxyl terminal truncate is in the residue 324-327[RSAK that crosses over SEQ ID NO:1, SEQ ID NO:219] random coil in, cause in the arginine place of the position 324 of SEQ ID NO:1, so that eliminate fully with the carboxyl terminal of HA2 effect.

PR/8HA1-2 construct (SEQ ID NO:9)For this construct, be by before the cysteine of the position 72 of SEQ ID NO:1, making the aminoterminal truncate, and behind the cysteine of the position 152 of SEQ ID NO:1, make the c-terminus truncate, and keep two (2) individual intrinsic disulfide bond (C72-C84 and the C107-C152 of SEQ ID NO:1).Use aforementioned crystal structure of quoting and MMD data base to identify three grades and secondary structure, amino terminal is to be positioned at the ring [KGI that connects two groups of different beta sheets with the carboxyl terminal truncate, the residue 62-64 of SEQ ID NO:1 and NASMHE (SEQ ID NO:220), the residue 285-290 of SEQ ID NO:1] in.Secondary structure around molecule truncate in these rings keeps, its follow-up tertiary structure that keeps ball head.

The amino terminal truncate is to cause in the lysine place of the position 62 of SEQ ID NO:1, so that comprise the residue 65-70 of the SEQ ID NO:1 of the β-thigh group that is positioned at away from film, β-thigh of APLQLG (SEQ ID NO:221) be kept perfectly, and than the preceding β-thigh [CRLK that is next near film, SEQ ID NO:216, residues 59-62 of SEQ ID NO:1] major part is eliminated.The carboxyl terminal truncate is to cause in the serine place of the position 284 of SEQ ID NO:1, so that the β-thigh [GIITS of long-range β-thigh group, SEQ ID NO:222, the residue 280-284 of SEQ ID NO:1] still keep complete or most of complete, and follow-up β-thigh [CNTK of near-end β-thigh group, SEQ ID NO:223, the residue 291-294 of SEQ ID NO:1] by complete or most of the elimination.Keep long-range β-thigh group, be the main determining factor on the functional domain border of selecting the HA1-2 construct.Long-range beta sheet (it comprises β-thigh of APLQLG (SEQ ID NO:221) [the residue 65-70 of SEQ ID NO:1], SYIVET (SEQ ID NO:224) [the residue 94-99 of SEQ ID NO:1] and GIITS (SEQ ID NO:222) [the residue 280-284 of SEQ ID NO:1]) act as, the stable stability secondary structure assembly that can guarantee tight functional domain structure.

PR/8HA1-3 construct (SEQ ID NO:10)For this construct, be by before the cysteine of the position 107 of SEQ ID NO:1, making the aminoterminal truncate, and behind the cysteine of the position 152 of SEQ ID NO:1, make the c-terminus truncate, and keep one (1) individual intrinsic disulfide bond (C107-C152 of SEQ ID NO:1).Use aforementioned crystal structure of quoting and MMDB data base to identify three grades and secondary structure, the amino terminal truncate is that the agedoite in the position 101 of SEQ ID NO:1 causes, cause the alpha-helix [NIAGWLLG before being next to, SEQ ID NO:225, the residue 73-80 of SEQ ID NO:1] and β-thigh [SYIVET, SEQ ID NO:224, the residue 94-99 of SEQ ID NO:1] be eliminated fully, keep follow-up β-thigh [PGDFI simultaneously, SEQ ID NO:226, the residue 109-113 of SEQ ID NO:1], and gang NSENGICY, the residue 101-108 of SEQ ID NO:227[SEQ ID NO:1], it comprises the intrinsic cysteine of the position 107 that is positioned at SEQ ID NO:1.

The carboxyl terminal truncate is that the destination county in nearly β-thigh [AFALSRGF, SEQ ID NO:228, the residue 270-277 of SEQ ID NO:1] causes.Last aminoacid of this strand (position 277 of SEQ ID NO:1) is phenylalanine, and it is eliminated and is unlikely and exposes this hydrophobic residue.The carboxyl terminal truncate is to cause in the glycine place of the position 276 of SEQ ID NO:1, and they are between two secondary structures.β-thigh [AFALSRG before this position truncate keeps being next to, SEQ ID NO:229, the residue 270-276 of SEQ ID NO:1] most of complete, and eliminate follow-up β-thigh [GIITS fully, SEQ ID NO:222, the residue 280-284 of SEQ ID NO:1].

The description on the functional domain border of VN04HA constructSelected boundary function territory be in following sequence (SEQ ID NO:2), emphasize as follows: HA1-1The border is to add single underscore (E50-K323 of SEQ ID NO:2);

The border is to add double underline (G62-E284 of SEQ ID NO:2); HA1-3The border is to add thick underline (N103-G276 of SEQ ID NO:2).The detailed description in each subunit design and boundary function territory sees also hereinafter.

60

MEKIVLLFAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDIL EKKHNGKLCDL

120

D

LRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPV NDLCYPGDFNDYEELKHL

180

LSRINHFEKIQIIPKSSWSSHEASLGVSSACPYQGKSSFFRNVVWLIKKNSTYPTIKRSY

240

NNTNQEDLLVLWGIHHPNDAAEQTKLYQNPTTYISVGTSTLNQRLVPRIATRSKVNGQSG

300

RMEFFWTILKPNDAINFESNGNFIAPEYAY KIVKKGDS

LEYGNCNTKCQTPMGA

360

INSSMPFHNIHPLTIGE CPKYVKSNRLVLATGLRNSPQRERRRKKRGLFGAIAGFIEGGW

420

QGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLE

480

RRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELG

540

NGCFEFYHKCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGIYQILSIYSTVA

565

SSLALAIMVAGLSLWMCSNGSLQCR(SEQ ID NO：2)

VN04 HA1-1 construct (SEQ ID NO:11)For this construct, be by before the cysteine of the position 58 of SEQ IDNO:2, making the aminoterminal truncate, and behind the cysteine of the position 318 of SEQ ID NO:2, make the c-terminus truncate, and keep four (4) individual intrinsic disulfide bond.The disulfide bond that is kept lists as follows: the C58-C290 of SEQ ID NO:2; C71-C83; C106-C151 and C294-C318.Use aforementioned crystal structure of quoting and molecular model to make data base (MMDB) and identify three grades and secondary structure, the amino terminal truncate is the glutamic acid that is positioned at the position 50 of SEQ ID NO:2, so make the β-thigh [THAQDI before being next to, SEQ ID NO:230, the residue 43-48 of SEQ ID NO:2] eliminated fully, and follow-up thigh [EKKHNG, SEQ ID NO:231, the residue 50-55 of SEQ ID NO:2], and by the formed β of the residue 56-61 of SEQ ID NO:2-thigh still keeps complete.On structure, comprise the residue 50-55[EKKHNG of SEQ ID NO:2, SEQ ID NO:231] random peptide, the β-thigh of film-long-range beta sheet is finished in emulation, so that this functional domain structure is kept perfectly.The carboxyl terminal truncate is near the residue 323-326[KSNR of SEQ ID NO:2, SEQ ID NO:233] ring zone (its be connected on the cysteine of position 318 of SEQID NO:2 after) in cause.This intra-annular truncate keeps, wherein by the residue 319-322[PKYV of SEQID NO:2, SEQ ID NO:234] formed β-thigh before being next to is still complete, and follow-up β-thigh [LVLATG, SEQ ID NO:235, the residue 327-332 of SEQ ID NO:2] secondary structure that is eliminated fully.

VN04HA1-2 construct (SEQ ID NO:12)For this construct, be by before the cysteine of the position 71 of SEQ IDNO:2, making the aminoterminal truncate, and behind the cysteine of the position 151 of SEQ ID NO:2, make the c-terminus truncate, and keep two (2) individual intrinsic disulfide bond (C71-C83 and the C106-C151 of SEQ ID NO:1).Use aforementioned crystal structure of quoting and molecular model to make data base (MMDB) and identify three grades and secondary structure, the amino terminal truncate is the glycine that is positioned at the position 62 of SEQ ID NO:2, so make the residue 64-68[KPLIL of the follow-up SEQ of comprising ID NO:2, SEQ ID NO:236] β-thigh still complete, and the β-thigh [KLCDLD before being next to, SEQ ID NO:232, the residue 56-61 of SEQ IDNO:2] be eliminated fully.

For keeping secondary structure, the carboxyl terminal truncate is to cause in the ring zone of the glutamic acid of the position 284 of SEQ ID NO:2, so that the β-thigh [TIMKS before being next to, SEQ ID NO:237, the residue 279-283 of SEQ ID NO:2] still complete, and follow-up β-thigh [YGNCN, SEQ ID NO:238, the residue 287-291 of SEQ ID NO:2] is eliminated fully.

VN04HA1-3 construct (SEQ ID NO:13)For this construct, be by before the cysteine of the position 106 of SEQ IDNO:2, making the aminoterminal truncate, and behind the cysteine of the position 151 of SEQ ID NO:2, make the c-terminus truncate, and keep one (1) individual intrinsic disulfide bond (C106-C151 of SEQ ID NO:2).

Use aforementioned crystal structure of quoting and molecular model to make data base (MMDB) and identify three grades and secondary structure, the amino terminal truncate is the agedoite that is positioned at the position 103 of SEQ ID NO:2, so make the β-thigh [SYIVEK before being next to, SEQ ID NO:239, the residue 93-98 of SEQ ID NO:2] be eliminated fully, and follow-up β-thigh [PGDFN, SEQ ID NO:240, the residue 108-112 of SEQ ID NO:2] keeps all or major part is complete.For keeping secondary structure, the carboxyl terminal truncate is to cause in the random coil zone of the glycine of the position 276 of SEQ ID NO:2, so that by the residue 102-105[KGDS of SEQ ID NO:2, SEQ ID NO:241] formed β-thigh before being next to still keeps whole or major part complete, β-thigh [EYAYKIVK before being next to, SEQ ID NO:242, the residue 267-274 of SEQ ID NO:2] be retained fully, and follow-up β-thigh [TIMKS, SEQ ID NO:237, the residue 279-283 of SEQ ID NO:2] secondary structure that is eliminated fully.

The description on the functional domain border of IND05HA constructIt is that sequence with reference A/ Vietnam/1203/2004 structure is calibrated to the basis that A/ Indonesia/5/2005 construct is described.Selected boundary function territory be in following sequence (SEQ ID NO:3), emphasize as follows: HA1-1The border is to add single underscore (E50-K323 of SEQ ID NO:3);

The border is to add double underline (G62-E284 of SEQ ID NO:3); HA1-3The border is to add thick underline (N103-G276 of SEQ ID NO:3).The detailed description in each subunit design and boundary function territory sees also hereinafter.

60

MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDIL EKTHNGKLCDL

120

D

LRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPT NDLCYPGSFNDYEELKHL

180

LSRINHFEKIQIIPKSSWSDHEASSGVSSACPYLGSPSFFRNVVWLIKKNSTYPTIKKSY

240

NNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISIGTSTLNQRLVPKIATRSKVNGQSG

300

RMEFFWTILKPNDAINFESNGNFIAPEYAY KIVKKGDS

LEYGNCNTKCQTPMGA

360

INSSMPFHNIHPLTIGE CPKYVKSNRLVLATGLRNSPQRESRRKKRGLFGAIAGFIEGGW

420

QGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLE

480

RRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELG

540

NGCFEFYHKCDNECMESIRNGTYNYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVA

560

SSLALAIMMAGLSLWMCSNGSLQCRICI(SEQ ID NO：3)

IND05 HA1-1 construct (SEQ ID NO:14)For this construct, be by before the cysteine of the position 58 of SEQ IDNO:3, making the aminoterminal truncate, and behind the cysteine of the position 318 of SEQ ID NO:3, make the c-terminus truncate, and keep four (4) individual intrinsic disulfide bond.The disulfide bond that is kept lists as follows: the C58-C291 of SEQ ID NO:3; C71-C83; C106-C151 and C294-C318.Use aforementioned crystal structure of quoting and molecular model to make data base (MMDB) and identify three grades and secondary structure, the amino terminal truncate is the glutamic acid that is positioned at the position 50 of SEQ ID NO:3, so make the β-thigh [THAQDI before being next to, SEQ ID NO:243, the residue 43-48 of SEQ ID NO:3] eliminated fully, and follow-up thigh [EKTHNG, SEQ ID NO:244, the residue 50-55 of SEQ ID NO:3], and still keep complete by the formed β of the residue 56-61 of SEQ ID NO:3-thigh [KLCDLD, SEQ ID NO:245].On structure, comprise the residue 50-55[EKTHNG of SEQ ID NO:3, SEQ ID NO:244] random peptide, it finishes the β-thigh of film-long-range beta sheet emulation, so that this functional domain structure is kept perfectly.The carboxyl terminal truncate is near the residue 323-326[KSNR of SEQ ID NO:3, SEQ ID NO:246] ring zone (its be connected on the cysteine of position 318 of SEQ ID NO:3 after) in cause.This intra-annular truncate, keep wherein residue 319-322[PKYV by SEQ ID NO:3, SEQ ID NO:247] formed β-thigh before being next to is still complete, and follow-up β-thigh [LVLATG, SEQ ID NO:248, the residue 327-332 of SEQ ID NO:3] secondary structure that is eliminated fully.

IND05 HA1-2 construct (SEQ ID NO:15)For this construct, be by before the cysteine of the position 71 of SEQ IDNO:3, making the aminoterminal truncate, and behind the cysteine of the position 151 of SEQ ID NO:3, make the c-terminus truncate, and keep two (2) individual intrinsic disulfide bond (C71-C83 and the C106-C151 of SEQ ID NO:3).Use aforementioned crystal structure of quoting and molecular model to make data base (MMDB) and identify three grades and secondary structure, the amino terminal truncate is the glycine that is positioned at the position 62 of SEQ ID NO:3, so make the residue 64-68[KPLIL of the follow-up SEQ of comprising ID NO:3, SEQ ID NO:249] β-thigh still complete, and the β-thigh [KLCDLD before being next to, SEQ ID NO:245, the residue 56-61 of SEQ IDNO:3] be eliminated fully.For keeping secondary structure, the carboxyl terminal truncate is that the glutamic acid in the position 284 of SEQ IDNO:3 (is positioned at [ELE, the residue 284-286 of SEQ ID NO:3] in) the ring zone in cause, so that the β-thigh [AIMKS before being next to, SEQ ID NO:250, the residue 279-283 of SEQ ID NO:3] still keep all or part of complete, and follow-up β-thigh [YGNCN, SEQ ID NO:251, the residue 287-291 of SEQ ID NO:3] be eliminated fully.

IND05 HA1-3 construct (SEQ ID NO:16)For this construct, be by before the cysteine of the position 106 of SEQ IDNO:3, making the aminoterminal truncate, and behind the cysteine of the position 151 of SEQ ID NO:3, make the c-terminus truncate, and keep one (1) individual intrinsic disulfide bond (C106-C151 of SEQ ID NO:3).Use aforementioned crystal structure of quoting and molecular model to make data base (MMDB) and identify three grades and secondary structure, the amino terminal truncate is the agedoite that is positioned at the position 103 of SEQ ID NO:3, so make the β-thigh [SYIVEK before being next to, SEQ ID NO:252, the residue 93-98 of SEQ ID NO:3] be eliminated fully, and follow-up β-thigh sequence [PGSFN, SEQ ID NO:253, the residue 108-112 of SEQ ID NO:3] keeps all or major part is complete.For keeping secondary structure, the carboxyl terminal truncate is to cause in the random coil zone of the glycine of the position 276 of SEQ ID NO:3, so that the β-thigh [EYAYKIVK before being next to, SEQ ID NO:254, the residue 267-274 of SEQ ID NO:3] be retained fully, and follow-up β-thigh [AIMKS, SEQ ID NO:250, the residue 279-283 of SEQ ID NO:3] is eliminated fully.

The description on the functional domain border of New Caledonia HA constructIt is that sequence with reference A/ Puerto Rico/8/34 structure is calibrated to the basis that A/ New Caledonia/20/199 construct is described.Selected boundary function territory be in following sequence (SEQ ID NO:4), emphasize as follows: HA1-1The border is to add single underscore (S53-R324 of SEQ ID NO:4);

The border is to add double underline (K62-S284 of SEQ ID NO:4); HA1-3The border is to add thick underline (N101-G276 of SEQ ID NO:4).The detailed description in each subunit design and boundary function territory sees also hereinafter.

60

MKAKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLED SHNGKLCL

120

L

QLGNCSVAGWILGNPECELLISKESWSYIVETP NPENGTCYPGYFADYEELRE

180

QLSSVSSFERFEIFPKESSWPNHTVTGVSASCSHNGKSSFYRNLLWLTGKNGLYPNLSKS

240

YVNNKEKEVLVLWGVHHPPNIGNQRALYHTENAYVSVVSSHYSRRFTPEIAKRPKVRDQE

300

GRINYYWTLLEPGDTIIFEANGNLIAPWYA FALSRGFG

NAPMDECDAKCQTPQG

360

AINSSLPFQNVHPVTIGE CPKYVRSAKLRMVTGLRNIPSIQSRGLFGAIAGFIEGGWTGM

420

VDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQFTAVGKEFNKLERRM

480

ENLNKKVDDGFLDIWTYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGC

540

FEFYHKCNNECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQILAIYSTVASSL

565

VLLVSLGAISFWMCSNGSLQCRICI(SEQ ID NO：4)

New Caledonia HA1-1 construct design (SEQ ID NO:17)For this construct, be by before the cysteine of the position 59 of SEQ ID NO:4, making the aminoterminal truncate, and behind the cysteine of the position 319 of SEQ ID NO:4, make the c-terminus truncate, and keep four (4) individual intrinsic disulfide bond.The disulfide bond that is kept lists as follows: the C59-C291 of SEQ ID NO:4; C72-C84; C107-C152 and C295-C319.Use reference crystal structure and MMDB to check secondary structure, the amino terminal truncate is that the serine in the position 53 of SEQ ID NO:4 causes, so make the β-thigh [THSVNLLED before being next to, SEQ ID NO:255, the residue 44-52 of SEQ ID NO:4] eliminated fully, and follow-up β-thigh SHNGKL, the residue 53-58 of SEQ ID NO:256[SEQ ID NO:4], and APLQLG, the residue 65-70 of SEQ IDNO:257[SEQ ID NO:4] still reservation is all complete.Be somebody's turn to do the amino terminal truncate in random coil sequence [SHNGKL, SEQ ID NO:256, the residue 53-58 of SEQ ID NO:4], the secondary structure major part be kept perfectly, because its simulation makes the complete β-thigh of beta sheet.The carboxyl terminal truncate is in random coil sequence [RSAK, SEQ ID NO:258, the residue 324-327 of SEQ ID NO:4] in the arginine (its be connected on the cysteine of position 319 of SEQ ID NO:4 after) of position 324 of SEQ ID NO:4 locate to cause.Truncate in this random coil, be by making the β-thigh [PKYV before being next to, SEQ ID NO:259, the residue 320-323 of SEQ ID NO:4] keep all complete, and follow-up β-thigh [LRMVTG, SEQ ID NO:260, the residue 328-333 of SEQ ID NO:4] be eliminated fully, and keep this secondary structure.

New Caledonia HA1-2 construct design (SEQ ID NO:18)For this construct, be by before the cysteine of the position 72 of SEQ ID NO:4, making the aminoterminal truncate, and behind the cysteine of the position 152 of SEQ ID NO:4, make the c-terminus truncate, and keep two (2) individual intrinsic disulfide bond (C72-C84 of SEQ IDNO:4 and C107-C152).Use reference crystal structure and MMDB to check secondary structure, the amino terminal truncate is that the lysine in the position 62 of SEQ ID NO:4 causes, so make and comprise [SHNGKLCLLKGI, SEQ ID NO:261, the residue 53-64 of SEQ ID NO:4] random coil sequence part or major part be removed, β-thigh [THSVNLLED before being next to, SEQ ID NO:255, the residue 44-52 of SEQ ID NO:4] eliminated fully, and follow-up β-thigh [APLQLG, SEQ ID NO:257, the residue 65-70 of SEQ ID NO:4] still reservation is all complete.The carboxyl terminal truncate is to cause in the serine place of the position 284 of SEQ IDNO:4, so that the β-thigh [SGIITS before being next to, SEQ ID NO:262, the residue 279-284 of SEQ ID NO:4] still reservation is all complete, and follow-up random coil [NAPMDECDA, SEQ ID NO:263, the residue 285-293 of SEQ ID NO:4] fully or major part be eliminated.

New Caledonia HA1-3 construct design (SEQ ID NO:19)For this construct, be by before the cysteine of the position 107 of SEQ ID NO:4, making the aminoterminal truncate, and behind the cysteine of the position 152 of SEQ ID NO:4, make the c-terminus truncate, and keep one (1) individual intrinsic disulfide bond (C107-C152 of SEQ IDNO:2).Use reference crystal structure and MMDB to check secondary structure, the amino terminal truncate is that the agedoite in the position 101 of SEQ ID NO:4 causes, so that the β-thigh [SYIVET before being next to, SEQ ID NO:264, the residue 94-99 of SEQ ID NO:4] eliminated fully, and that follow-up β-thigh [PGYFA, SEQ ID NO:265, the residue 109-113 of SEQ ID NO:4] still keeps is all complete.For keeping secondary structure, the carboxyl terminal truncate is that the glycine in the position 276 of SEQ ID NO:4 causes, so that β-thigh sequence [YAFALSRGF, SEQ ID NO:266, the residue 269-277 of SEQ ID NO:4] be retained fully, and follow-up β-thigh sequence [SGIITS, SEQ ID NO:262, the residue 279-284 of SEQ IDNO:4] is eliminated.

The description on the functional domain border of A/ winconsin/67/2005HA constructIt is that sequence with reference A/X31 structure is calibrated to the basis that A/ winconsin/67/2005 construct is described.Selected boundary function territory be in following sequence (SEQ ID NO:6), emphasize as follows: HA1-1The border is to add single underscore (Q44-K310 of SEQ ID NO:6);

The border is to add double underline (S54-D271 of SEQ ID NO:6); HA1-3The border is to add thick underline (S95-G263 of SEQ ID NO:6).The detailed description in each subunit design and boundary function territory sees also hereinafter.

60

QKLPGNDNSTATLCLGHHAVPNGTIVKTITNDQIEVTNATELV QSSSTGGIC

LD

120

GENCTLIDALLGDPQCDGFQNKKWDLFVERSKAY SNCYPYDVPDYASLRSLVASSGTLEF

180

NDESFNWTGVTQNGTSSACKRRSNNSFFSRLNWLTHLKFKYPALNVTMPNNEKFDKLYIW

240

GVHHPGTDNDQIFLHAQASGRITVSTKRSQQTVIPNIGSRPRIRNIPSRISIYWTIVKPG

300

DILLINSTGNLIAPRGY FKIRSGKS

APIGKCNSECITPNGSIPNDKPFQNVNRI

329

TYGA CPRYVKQNTLKLATGMRNVPEKQTR(SEQ ID NO：6)

A/ winconsin/67/2005HA1-1 construct design (SEQ ID NO:20)For this construct, be by before the cysteine of the position 52 of SEQ ID NO:6, making the aminoterminal truncate, and behind the cysteine of the position 305 of SEQ IDNO:6, make the c-terminus truncate, and keep four (4) individual intrinsic disulfide bond.The disulfide bond that is kept lists as follows: the C52-C277 of SEQ ID NO:6; C64-C76; C97-C139 and C281-C305.Use reference crystal structure and MMDB to check secondary structure, the amino terminal truncate is to cause in the glutamic acid of the position 44 of SEQ ID NO:6 (it is next to residue 42 and 43[LV by SEQ ID NO:6] formed tight turn after), so that the β-thigh [TNATE before being next to, SEQ IDNO:267, the residue 37-41 of SEQ ID NO:6] eliminated fully, and by the 44-46 of residue QSS[SEQ ID NO:6] whole short β-thigh of forming is retained.The carboxyl terminal truncate is in random coil sequence [PRYVK, SEQ ID NO:268, the residue 306-310 of SEQ ID NO:6] cause in (its be connected on the cysteine of position 305 of SEQ ID NO:6 after), so that should be removed fully with the c-terminus afterbody of HA2 effect.Though carboxyl terminal sequence [PRYVK, SEQ ID NO:268, the residue 306-310 of SEQ ID NO:6] be defined as random coil, but in textural its is to be provided as further to stablize, residue 44-46[QSS by SEQ IDNO:6] and the residue 293-297[PFQNV of SEQ ID NO:6, SEQ ID NO:269] the extra β-thigh of the beta sheet that defined.This group β-flap comprises amino terminal and the carboxyl terminal that is arranged in a stable secondary structure assembly, to guarantee tight functional domain structure.

A/ winconsin/67/2005HA1-2 construct design (SEQ ID NO:21)For this construct, be by before the cysteine of the position 64 of SEQ ID NO:6, making the aminoterminal truncate, and behind the cysteine of the position 139 of SEQ IDNO:6, make the c-terminus truncate, and keep two (2) individual intrinsic disulfide bond (C64-C76 and the C97-C139 of SEQ ID NO:6).Use reference crystal structure and MMDB to check secondary structure, the amino terminal truncate is to cause in the ring that connects two groups of different beta sheets.The amino terminal truncate is to cause in the serine place of the position 54 of SEQ ID NO:6, so that the residue 57-62[QILDGE that comprises SEQ IDNO:6 of film-long-range β-thigh group, SEQ ID NO:270] β-thigh still keep complete fully, and film-near-end β-thigh [GGICD, SEQ ID NO:271, the residue 49-53 of SEQ ID NO:6] eliminated fully.The carboxyl terminal truncate is that the aspartic acid in the position 271 of SEQ ID NO:6 causes.Truncate in this position keeps so that film-long-range β-thigh [SSIMRS, SEQ ID NO:272, the residue 265-270 of SEQ ID NO:6] still all complete, and the secondary structure that film-near-end β-thigh [APIGK, SEQ ID NO:273, the residue 272-276 of SEQ ID NO:6] is eliminated.Comprising film-long-range beta sheet of β-thigh QILDGE (SEQ ID NO:270) [the residue 57-62 of SEQ ID NO:6], LFVER (SEQ ID NO:274) [the residue 86-90 of SEQ ID NO:6] and SSIMRS (SEQ ID.NO:272) [the residue 265-270 of SEQ ID NO:6], is to be provided as the stability secondary structure of guaranteeing tight functional domain structure.

A/ winconsin/67/2005HA1-3 construct design (SEQ ID NO:22)For this construct, be by before the cysteine of the position 97 of SEQ ID NO:6, making the aminoterminal truncate, and behind the cysteine of the position 139 of SEQ IDNO:6, make the c-terminus truncate, and keep one (1) individual intrinsic disulfide bond (C97-C139 of SEQ ID NO:2).Use reference crystal structure and MMDB to check secondary structure, the amino terminal truncate is to cause in the serine place of the position 95 of SEQ ID NO:6, so that the alpha-helix [TLIDALLG before being next to, SEQ ID NO:275, the residue 65-72 of SEQ ID NO:6] eliminated fully, and the serine of the position 95 of SEQ ID NO:6, and the agedoite of the position 96 of SEQ ID NO:6 is retained with the cysteine of the position 97 of SEQ ID NO:6.The carboxyl terminal truncate is in the random coil sequence, cause at the about glycine place of the position 263 of SEQ ID NO:6, so that this contains the residue 261-263[RSG of SEQ ID NO:6] the random coil sequence still keep all or part of complete, β-thigh [RGYFKI before being next to simultaneously, SEQ ID NO:276, the residue 255-260 of SEQ ID NO:6] all kept, and follow-up β-thigh [SSIMRS, SEQ ID NO:272, the residue 265-270 of SEQ ID NO:6] be eliminated fully.

Discuss

The proteic part ball head of influenza virus haemagglutinin (HA) functional domain can produce neutrality antibody (cloth Lander (Brand) and Shi Qier (Skehel), 1972; Eckert (Eckert), 1973; Jackson people such as (Jackson), 1979; Lu Si people such as (Russ), 1981), and this functional domain separately, chondritic is considered to constitute can be through being expressed in antibacterial with folding again, or express and oozy protein fragments from eucaryon host, exempt when making total length HA albumen the problem on the expression and purification that has faced thus.

Merge maximum HA construct (also being called " HA fragment "), include the most of or almost whole bulbous region in natural (wild type) HA1 header functionality territory in this paper to STF2, HA1-1 (SEQ ID NO:8,11,14,17,20).Natural HA1 fragment with from virion in limited albuminolysis the time, the HA1-1 construct that is disengaged closely similar (than the people such as (Bizeband) of nation now, 1995).Therefore, structural analysis and experimental data hint, the HA1-1 construct should effectively fold (no matter it is to make) in antibacterial or eukaryotic cell, and keeps stabile class natural structure.Equally, expect whole HA0, form by HA1 and HA2 but get rid of outside the signal peptide (17aa), be positioned at the outer part of HA molecule (SEQID NO:23) of viromembrane outside, be positioned at the internal peptide (12aa) of viromembrane and stride film functional domain (24aa), when being expressed in eucaryon host, should possess its native configurations, maybe when being expressed in antibacterial, can be in vitro through being folded into native state again.Two less HA fragment (HA1-2 (SEQ ID NO:9,12,15,18,21) with HA1-3 (SEQID NO:10,13,16,19,22)), can making the protein fragments that is become remove to stabilize (HA1-3), or the side chain of the hydrophobic residue that some are big exposes the mode of (HA1-2), the zone that truncate header functionality territory is long-range apart from receptor binding site.For this reason, satisfy experimental mutation effect importing HA1-2 and HA1-3 construct, called after HA1-2mut (SEQ ID NO:24,26,28,30,32) and HA1-3mut (SEQ ID NO:25,27,29,31,33).The replacement of carrying out in HA1-3mut (SEQ ID NO:25,27,29,31,33) construct is to import being positioned at the tool opposite charges residue that is still exposed by the HA1-3 truncate on relative strand." HA mut " is used for this paper and means, exist among the natural HA aminoacid by, do not betide the aminoacid replacement of this natural HA.The residue that is substituted can form salt bridge, and stablizes the structure that not so it may or fold poor (or folding completely).For example, in PR8, G105 is with the glutamic acid displacement, and Y115 replaces (SEQ ID NO:25) through lysine.The glutamic acid of electronegative property electric charge and the lysine effect of positively charged electric charge, enable pass is crossed and the short spiral 15-22[NSENEICY that is positioned at branch subcenter SEQ ID NO:25, SEQ ID NO:277], the electric charge-charge interaction of lysine 15 of N-end, stablize the N-end peptide 1-8 of SEQ ID NO:25.For the HA1-2 construct, truncate can cause exposing hydrophobic side chains, and it is not expected can reduce protein expression.Yet the residue that exposes may cause expressed proteinic gathering, or makes expressed protein unease.For avoiding assembling and increasing the stability of expressed molecule, then those are become the big hydrophobic residue that exposes, with more neutral aminoacid replacement, and produce HA1-2mut (SEQ ID NO:24,26,28,30,32).These substituent groups can make protein more comply with tough operation sequence, and/or have long-term stability.

Embodiment 2: the part of the natural hemagglutinin of design Type B influenza virus

Materials and methods

Design is for HA1-1,1-2 and the 1-3 ball head construct of B/Lee/40Structure in design influenza B HA vaccine is considered, be to described similar about A type influenza virus person, that is, must identify the functional domain border of HA ball head, so that flagellin-HA fusion rotein mass-energy is correctly folding, or correctly folding again and expose suitable antigenicity epitope., can't not obtain very definite X-ray crystal structure on the influenza B HA, the therefore difficult functional domain border of defining ball head clearly like influenza A.So, must be based on bioinformatics and structural model, predict influenza B HA pattern (Tong Dengren, 2004.J.Gen.Virl.85,3249-3259).These researchs are to use " knowledge is the basis " general plan, and it is dependent on the known structure that derives from Protein Data Bank, and the height sequence homology between the target unknown structure.Generally, this general plan is the most favourable to have at least 35% sequence homogeneity between known and target protein.

In the case of influenza B, immediate pattern is from A/ pig/Hong Kong/9/98 (SEQ ID NO:34) (24% homogeneity, PDB login password 1JSD) and A/Aichi/2/68 (SEQ ID NO:35) (21% homogeneity, PDB login password 1HGF).Though the sequence homogeneity between target Mode B/Lee/40HA (SEQ ID NO:36) and known template pattern, in fact be lower than desirable minima 35%, although but they's sequence difference (the total only 18% sequence homogeneity of H1, H3, H5 and H9), the proteinic function of influenza A HA and three grades of folding close similaritys, hint might use influenza A HA pattern successfully to predict influenza B HA structure.And although influenza C HEF (hemagglutinin-esterase-fusion) proteic folding and influenza A HA structural similarity are its sequence homogeneity even be lower than any A/B comparison.

Because the crystal structure of influenza C HEF is known (PDB accession number 1FLC, MMDB accession number 12663),, predict influenza B HA structure so Tong Dengren comprises the knowledge of structure homogeneity between C HEF and known influenza A HA albumen.People such as child at first will derive from the HEF sequence (SEQ ID NO:37) of C/ Johannesburg/1/66, use CLUSTALW (people such as Tang Pusheng with a kind of sequence (http://flu.lanl.gov) of the 15HA hypotype out of the ordinary that derives from A type influenza virus, 1994.Nucleic Acids Res 22,4673-4680) calibrate, and be calibrated to the basis with structure-announcement and compile collection of illustrative plates.

Catch and specify the conservative second structure characteristic, and between various and each hypotype, the variation that causes because of time and host type.They further calibrate the A/C collection of illustrative plates of amplification then, to the calibration of influenza B HA sequence (comprising Lee B//40).Use homology modelling technique construction Lee B//40 patterns of virgin (1999).

Letter speech, they at first connect structure with the main chain of target thing, connect structure with the main chain of template and match in institute's calibration region.With in the target thing with respect to the insertion fragment of template, the ring that is considered as having known end structure is handled.Expected structure is launched, comply with in the template insertion fragment with respect to the target thing.Connect structure model (child, 1997 by the main chain that uses effective Mondicaro ring-sampling method to make ring; People such as Liu, 1998).In case after main chain connect structure modelling model, just the side chain atom is connected.Because the head of HA molecule is tight, so can be limited for the space of setting side chain atom.Therefore, in this is analyzed, the side chain torsion angle is initialized to equals or be similar to person in the formwork structure.This consideration is particularly useful for avoiding the interchain conflict of each side in the molded structure.At last, complete-atomic model is passed through to use AMBER (prestige energy people such as (Weiner), 1986), carry out short distance energy minimization bout (1000 circulation), to disengage the more disadvantageous three-dimensional spatial chemistry optimization that acts on and make.By PROCHECK (Weir Lignum Rhamnellae people .1998.J.MolBiol.276,417-436) the quality of inspection target object model, and functional be to stop (substrate docking) by substrate to test this model substrates binding site and whether can comply with natural substrate analog sialyl lactose and (can like in A/ as we to know/observedly in 2/68 the HA crystal structure test to person's (this grade of prestige people .1988. nature 333,426-431)).Lee B/ of institute's emulation/40 models are at Ramachandran figure (Weir Lignum Rhamnellae people .1998.J.MolBiol.276, show 417-436) as if rationally, and when making sialic acid-2-3-lactose molecule stop substrate binding site into Lee B//40 models, show the conflict on the no solid space, represent that it has correct spatial chemistry.So, use the design of this Lee B//40 models guiding influenza B HA subunit vaccine.

Result and discussion

The description on the functional domain border of Lee B//40HA constructSelected boundary function territory be in following sequence, emphasize as follows: HA1-1The border is to add single underscore (T48-K340 of SEQ ID NO:36);

The border is to add double underline (K60-G299 of SEQ ID NO:36).The detailed description in each subunit design and boundary function territory sees also hereinafter.

60

MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPL TTTPTKSHFANL

120

GKLCPNCFNCTDLDVALGRPKCMGNTPSAKVSILHEVKPATSGCFPIMHDRTKIR

180

QLPNLLRGYENIRLSTSNVINTETAPGGPYKVGTSGSCPNVANGNGFFNTMAWVIPKDNN

240

KTAINPVTVEVPYICSEGEDQITVWGFHSDDKTQMERLYGDSNPQKFTSSANGVTTHYVS

300

QIGGFPNQTEDEGLKQSGRIVVDYMVQKPGKTGTIVYQRGILLPQKVWCASGR

S

360

LPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGN CPIWVKTPLKLANGTKYRPPAKLLKE

420

RGFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAADLKSTQEAINKITKNLNYLSELE

480

VKNLQRLSGAMNELHDEILELDEKVDDLRADTISSQIELAVLLSNEGIINSEDEHLLALE

540

RKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFNAGDFSLPTFDSLNITAASLND

584

DGLDNHTILLYYSTAASSLAVTLMIAIFIVYMVSRDNVSCSICL(SEQ.ID NO：36)

The coordinate of the B/Lee/40 model of institute's emulation can be obtained with the 1TX1 that accesses to your password in Protein Data Bank.The pdb shelves are by using VAST (carrier calibration search tools) to search (http://www.ncbi.nlm.nih.gov/Structure/VAST/vastsearch.html), converting MMDB (molecular model is made the data base) form to.Watch the model of 1TX1 structure then by Cn3D, and structure is stored with the MMDB form.Based on design influenza A HA1-1 and the employed same principle of HA1-2 construct,, accurately make the functional domain border of Lee B//40HA1-1 and HA1-2 by checking this model structure.Lee B//40HA1-1 (SEQ ID NO:38) comprises the long-range β-flap of spherical top, film that epitope is concentrated and is positioned at the extra β-interlayer of the long-range β of this film-flap below.The amino terminal of HA1-1 originates in the residue 48 of SEQ ID NO:36, and end at residue 340 (TTTPTK (SEQ ID NO:279) ...-... CPIWVK (SEQ ID NO:280)).B/Lee/40HA1-2 (SEQ ID NO:39) is by shifting out the design of β-interlayer from HA1-1.HA1-2 originates in the residue 60 of SEQ ID NO:36, and end at residue 299 (KGTQTR (SEQ ID NO:281) ...-... SKVIKG (SEQ ID NO:282)).For determining the border selection, also use other independent solution.Employed first method is the primary sequence calibration.A/ liked know/sequence and Lee B//40 (the SEQ ID NO:36) sequence of 2/68 (SEQ ID NO:35) calibrate.Calibrate out A/ and like to know/border of 2/68HA1-1 (SEQ ID NO:40) (residue 60-326QSSSTG (SEQ ID NO:283) ...-... CPKYVK (SEQ ID NO:284)) and border (the residue 72-287 of HA1-2 (SEQID NO:41); HRILDG (SEQ ID NO:285) ...-... SIMRSD (SEQID NO:286)) with being harmonious closely of Lee B//40, support the border selection of this uses institute phantom.Carry out the residue adjustment to avoid exposing the two ends of first hydrophobic residue and construct.Therefore, 3-dimension structure prediction and primary sequence calibration meet very much.

Employed second method is a secondary structure prediction.The functional domain border generally is to be arranged in ring or corner area, and can not invade such as a lot of in the secondary structure assemblies such as alpha-helix and β-flap, especially the center of alpha-helix bundle or β-flap.This condition is used for rechecking, selects, whether meet independent secondary structure prediction by the border that the 3-D structure of institute's emulation causes.Service routine PHD ( Http:// ca.expasy.org/tools→ Albuminous body is learned and the sequence analysis instrument→ Secondary and tertiary structure worker Tool→ Predicted protein matter) carry out secondary structure prediction.In HA1-2 carboxyl-terminal residue two with the overlapping person of short alpha-helix (5 aminoacid), all other border residues all fall within the ring zone, are expressed as rational border and select.PHD result lists as follows, and wherein AA is an aminoacid sequence; OBS_sec is observed secondary structure: H=spiral, E=extension flap, blank=other (ring); The secondary structure that PROF_sec:PROF predicted: the H=spiral, extend (flap) blank=other (ring), PROF=PROF: collection of illustrative plates network prediction Heidelberg; The credibility index (0=is low to moderate the 9=height) of Rel_sec:PROFsec prediction.

For simple expression, strong prediction by ' ^*' indicate; Time group of SUB_sec:PROFsec prediction has the bat of expection for all residues〉82%.Attention: be to use following symbol: L: be ring (its use subscript ' ') for this group; .: mean this residue and predict, because credibility is: Rel＜5; O_3_acc: viewed relative solvent accessibility (acc) is under 3 states: b=0-9%, i=9-36%, e=36-100%; The desired relative solvent accessibility of P_3_acc:PROF (acc) is under 3 states: b=0-9%, i=9-36%, e=36-100%; The credibility index (0=is low to moderate the 9=height) of Rel_acc:PROFacc prediction.

For simple expression, strong prediction is by ' * ' sign; Time group of SUB_sec:PROFsec prediction has the average relevant of expection for all residues〉0.69% (major event form).

Attention: be to use following symbol for this group:

I: be intermediate (it uses subscript ＂)

.: mean this residue and predict, because credibility is: Rel＜4.

....，....1....，....2....，....3....，....4....，....5

AA MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTT

OBS sec

PROF sec EEEEEEEEEE EEEEEEEE EEEEEE EEEEEEEEEE

Rel sec 90456542111257762677454037753444441276147765653203

SUB sec L..EEE......LLLL.EEE.E...LLL........LL..EEEEEE....

O 3 acc bbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbb

P 3 acc ee bbbbbbbbbeeb bbbbbbb eeee b bb eeebe b bbbe

Rel acc 33169997521111032778921201222233322135234021321012

SUB acc ...bbbbbb........bbbb................e..b.........

....，....6....，....7....，....8....，....9....，....10

AA PTKSHFANLKGTQTRGKLCPNCFNCTDLDVALGRPKCMGNTPSAKVSILH

OBS sec

PROF sec EEEE EEEEEE EEEE

Rel sec 67752200245344675222033211232342043234337875203787

SUB sec LLLL......L...LLL.......................LLLL...EEE

O 3 acc bbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbb

P 3 acc eee bbe eeeeeee eb bbbbbbbbb b b ee eeeeb bbb

Rel acc 23201011020002300210201031223343310100210221541265

SUB acc ..............................b.............eb..bb

....，....11.1.，....12.1.，....13.1.，....14.1.，....15.1

AA EVKPATSGCFPIMHDRTKIRQLPNLLRGYENIRLSTSNVINTETAPGGPY

OBS sec

PROF sec EE HHHHHHHHHHH EEEEE EE

Rel sec 50566441224778858887664221002304663021003444556531

SUB sec E.LLL......LLLLHHHHHHH..........EE..........LLLL..

O 3 acc bbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbb

P 3 acc b eeee bbbbb e b ebbebbee eeb eeeeee

Rel acc 12210110221001337264350314531332301200011120132011

SUB acc ................e.bi.b...be.......................

....，....16.1.，....17.1.，....18.1.，....19.1.，....20.1

AA KVGTSGSCPNVANGNGFFNTMAWVIPKDNNKTAINPVTVEVPYICSEGED

OBS sec

PROF sec HHHHHHHHHHH EEEEEE

Rel sec 24431001255662036787888752378864236641012244157874

SUB sec .........LLLL...HHHHHHHHH..LLLL...LL.........LLLL.

O 3 acc bbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbb

P 3 acc bbbbbbee ee bbbbbbb bbeeeee ee ebeb b beee

Rel acc 11111012110301026314921522122321112112322203203212

SUB acc ................b..bb..b..........................

....，....21.1.，....22.1.，....23.1.，....24.1.，....25.1

AA QITVWGFHSDDKTQMERLYGDSNPQKFTSSANGVTTHYVSQIGGFPNQTE

OBS sec

PROF sec EEEEEEEE HHHHHH EEEEE EEEEEEEE

Rel sec 47897532588742544320368853775200122343201275331035

SUB sec .EEEEE..LLLL..H......LLLL.EEE.............LL.....L

O 3acc bbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbb

P 3acc bbbbbbbbb eeee b eeeeeeb b b e e e ebee b e

Rel acc 19696993033223122210203213224011122211311001202001

SUB acc .bbbbbb.....................b.....................

....，....26.1.，....27.1.，....28.1.，....29.1.，....30.1

AA DEGLKQSGRIVVDYMVQKPGKTGTIVYQRGILLPQKVWCASGRSKVIKGS

OBS sec

PROF sec EEEEEEEEE EEEEEE EEE EEEEE EEEE

Rel sec 56754320377888983577247886434023022235520330245401

SUB sec LLLL.....EEEEEEE.LLL..EEEE...........EE.......E...

O 3acc bbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbb

P 3acc eee bbbbbbb bbbb ee b b bbbbbb bb b eeb bbe e

Rel acc 17211105270744553301222473010244520230221020211103

SUB acc .e.....b.b.bibbb.......ib.....bbb.................

....，....31.1.，....32.1.，....33.1.，....34.1.，....35.1

AA LPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTK

OBS sec

PROF sec HHH HHHHH HHHHH

Rel sec 32233333000113432246764334340342334133220003551573

SUB sec ...................LLL......................HH.LL.

O 3acc bbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbb

P 3acc e e e bb ee b bbbebbe be bb bbbbb

Rel acc 10010011112110110111101210102102111021712452461321

SUB acc ......................................b..bb.bb....

....，....36.1.，....37.1.，....38.1.，....39.1.，....40.1

AA YRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAADLK

OBS sec

PROF sec EEEEHHHH EEEEE H

Rel sec 56853211324203110113404531100120021023564325530623

SUB sec LLLL...................L..............LL...EE..L..

O 3 acc bbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbb

P 3 acc b eee be bbbbbbbbbbbbbb bbbbbbbbbbbbbb bbbbbbb e

Rel acc 00011411202355999999623520456353043010011315264032

SUB acc .....e......bbbbbbbbb..b..bbb.b..b.........b.bb...

....，....41.1.，....42.1.，....43.1.，....44.1.，....45.1

AA STQEAINKITKNLNYLSELEVKNLQRLSGAMNELHDEILELDEKVDDLRA

OBS sec

PROF sec HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH HHH

Rel sec 47888989873212477733101234446677898888878756210111

SUB sec .HHHHHHHHH.....HHH..........HHHHHHHHHHHHHHHH......

O 3 acc bbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbb

P 3 acc bbbebbeebbe b bbee beebeeb ebbeebbe beeb eebee

Rel acc 28049825725342322312063124534063444467553254223101

SUB acc .b.ebb.eb.e.b........e...eb.e.b.ebbeibee..ee......

....，....46.1.，....47.1.，....48.1.，....49.1.，....50.1

AA DTISSQIELAVLLSNEGIINSEDEHLLALERKLKKMLGPSAVEIGNGCFE

OBS sec

PROF sec HHHHHHHHHHHHHH HHHHHHHHHHHHHHHHHHHH EEE

Rel sec 03645477777753464114550578879888887664424540663252

SUB sec ..H.H.HHHHHHH..L....LL.HHHHHHHHHHHHHH....H..LL..E.

O 3 acc bbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbb

P 3 acc bbbb bb bbbbb eebb e e b eb e b e b e bee eebbbb

Rel acc 04450592968651122001021323439443944173428232012340

SUB acc .bbb.bb.bbbbb.............i.bie.bie.b.e.b.......b.

....，....51.1.，....52.1.，....53.1.，....54.1.，....55.1

TKHKCNQTCLDRIAAGTFNAGDFSLPTFDSLNITAASLNDDGLDNHTILL(SEQIDNO：

AA 36)

OBS sec

PROF sec EEEE HHHHHHHH HHHH EEEEEEEEE EEEEE

Rel sec 22003402344311355466523223103101002476217776303665

SUB sec ...............LL.LLL...............EE..LLLL...EEE

O 3 acc bbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbb

P 3 acc b beb e bbbbb bbbb ee eebbe beb bbeb eeeeeeb bbb

Rel acc 42120312620271012211330320211224131235303222133868

SUB acc b.......b...b..................e.....b.........bbb

....，....56.1.，....57.1.，....58.1.(SEQ ID NO：36)

AA YYSTAASSLAVTLMIAIFIVYMVSRDNVSCSICL

OBS sec

PROF sec EEEHHHHHHHHHHHHHHHHHHH EEEEEEE

Rel sec 4310021478888888776321006740346860

SUB sec ........HHHHHHHHHHH.....LL....EEE.

O 3 acc bbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbb

P 3 acc bbbbbbbbbbbbbbbbbbbbbbbee eb b bbb

Rel acc 6362366679999996998517101010152400

SUB acc b.b..bbbbbbbbbbbbbbb.b.......b.b..

Employed the third instrument is a hydrophobicity analysis.Generally, proteinic structural core is tended to, and is a kind of hydrophobicity clustering that extends to flank with the hydrophilic residue.So the border of being predicted should not be positioned at hydrophobic core entreats in the heart, but should be arranged in the both wings hydrophilic region.Use ProtScale (http://ca.expasy.org/tools → Primary Structure Analysis→ ProtScale) draw the hydrophobicity of Lee B//40HA sequence (SEQ ID NO:36).Lee's B//40 sequences (SEQ ID NO:36) hydrophobicity analysis determines that selected border is to be positioned at this proteinic hydrophilic region (Fig. 2).Be used for the hydrophobicity intensity of this figure, be shown in following table:

Sequence length: 584

For identifying other influenza B molecule, for example B/ Malaysia/2506/2004 (SEQ ID NO:42), B/ Ohio/1/2005 (SEQ ID NO:43), B/ Victoria/2/87 kind are (SEQ ID NO:787) and B/ Shanghai/361/2002 (SEQ ID NO:44) and B/ chevron/16/88 kind is (SEQ ID NO:213), HA 1-1 and HA1-2 functional domain border, then each HA sequence and Lee B//40HA sequence (SEQ ID NO:36) are calibrated.The result points out that influenza B HA albumen is more conservative compared to influenza A HA albumen, particularly in the functional domain border sequence.Therefore, be used in Lee B//40 identify the border, carry out the direct border of other Type B strains of influenza viruses and select.Gained functional domain border for each strain is summarized in down.

B/ Malaysia/2506/2004:HA1-1 44-337 (SEQ ID NO:45) (TTTPTK (SEQ IDNO:287)-CPIWVK (SEQ ID NO:288)) HA1-2 56-296 (SEQ ID NO:46) (KGTETR (SEQ ID NO:289)-SKVIKG (SEQ ID NO:290))

B/ Ohio/1/2005:HA1-1 33-326 (SEQ ID NO:47) (TTTPTK (SEQ ID NO:291)-CPIWVK (SEQ ID NO:292)) HA1-2 45-285 (SEQ ID NO:48) (KGTKTR (SEQ ID NO:293)-SKVIKG (SEQ ID NO:294))

B/ Shanghai/361/2002:HA1-1 33-325 (SEQ ID NO:49) (TTTPIK (SEQ ID NO:295)-CPIWVK (SEQ ID NO:296)) HA1-2 45-284 (SEQ ID NO:50) (KGTRTR (SEQ ID NO:297)-SKVIKG (SEQ ID NO:298))

50

BMa1 ----IVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTT

BOhi ---------------DRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTT

BLee MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTT

BSha ---------------DRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTT

***********************************

100

BMal PTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGNIPSARVSILH

BOhi PTKSHFANLKGTKTRGKLCPKCLNCTDLDVALGRPKCTGNIPSAEVSILH

BLee PTKSHFANLKGTQTRGKLCPNCFNCTDLDVALGRPKCMGNTPSAKVSILH

BSha PIKSHFANLKGTRTRGKLCPDCLNCTDLDVALGRPMCVGTTPSAKASILH

* **********.*******.*:************ * *.***..****

150

BMal EVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGSY

BOhi EVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAEKAPGGPY

BLee EVKPATSGCFPIMHDRTKIRQLPNLLRGYENIRLSTSNVINTETAPGGPY

BSha EVRPVTSGCFPIMHDRTKIRQLPNLLRGYENIRLSTQNVIDAEKALGGPY

**:*.*************************:********::*.* **.*

200

BMal KIGTSGSCPNVTNGNGFFATMAWAVPKNDNNKTATNSLTIEVPYICTEGE

BOhi KIGTSGSCPNVTNGNGFFATMAWAVPKNDNNKTATNSLTIEVPYICTEGE

BLee KVGTSGSCPNVANGNGFFNTMAWVIPK-DNNKTAINPVTVEVPYICSEGE

BSha RLGTSGSCPNATSKSGFFATMAWAVPK-DNNKNATNPLTVEVPYICTEGE

::********.:..*** ****.:** ****.* *.:*：******:***

250

BMal DQITVWGFHSDNEAQMAKLYGDSKPQKFTSSANGVTTHYVSQIGGFPNQT

BOhi DQITIWGFHSDSETQMAKLYGDSKPQKFTSSANGVTTHYVSQIGGFPNQT

BLee DQITVWGFHSDDKTQMERLYGDSNPQKFTSSANGVTTHYVSQIGGFPNQT

BSha DQITVWGFHSDDKTQMKNLYGDSNPQKFTSSANGVTTHYVSQIGGFPDQT

****:******.::** .*****:***********************:**

300

BMal EDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKG

BOhi EDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKG

BLee EDEGLKQSGRIVVDYMVQKPGKTGTIVYQRGILLPQKVWCASGRSKVIKG

BSha EDGGLPQSGRIVVDYMVQKPGKTGTIVYQRGVLLPQKVWCASGRSKVIKG

** ** *************.******.****:******************

350

BMal SLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGT

BOhi SLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGT

BLee SLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGT

BSha SLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGHCPIWVKTPLKLANGT

**********************************:***************

400

BMal KYRPPAKLLKER--------------------------------------

(SEQ ID NO：42)

BOhi KYRPPAKLLKERGF------------------------------------

(SEQ ID NO：43)

BLee KYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAADL

(SEQ ID NO：36)

BSha KYRP----------------------------------------------

(SEQ ID NO：44)

****

B/Lee/40HA1-1 construct (SEQ ID NO:38)For this construct, be by before the cysteine of the position 69 of SEQ IDNO:36, making the aminoterminal truncate, and behind the cysteine of position 335, make the c-terminus truncate, and keep five (5) individual intrinsic disulfide bond.Two sulfur pairings of described likelihood are as follows: the C69 of SEQID NO:36 and C72, C75 and C87, C109 and C158, C195 and C289, C309 and C335.Use simulation architecture and MMBD to check secondary structure, the amino terminal truncate be in the threonine of the position 48 of SEQ IDNO:36 (its be next to β-thigh that the proline-4 6 by SEQ ID NO:36 imports twist together connect leucine 47 subsequently after) cause, so that the β-thigh [TGVI before being next to, SEQ ID NO:299, the residue 42-45 of SEQ ID NO:36] eliminated fully, and the whole short β-thigh that is formed by residue TTTP (SEQ ID NO:300) [48-51 of SEQ ID NO:36] is retained.The carboxyl terminal truncate is that the end of the β-thigh PIWV (SEQ ID NO:301) [the residue 336-340 of SEQ ID NO:36] behind the cysteine of following position 335 causes, so that should be removed fully with the c-terminus afterbody of HA2 effect.Comprise six β-thighs of the terminal β of C--thigh, form by β-thigh PIWV (SEQ ID NO:301) [the residue 336-340 of SEQ ID NO:36], PYYTG (SEQ ID NO:302) [residue 322 to 326 of SEQ ID NO:36] and TTTP (SEQ ID NO:300) [the residue 48-51 of SEQ ID NO:36] (below beta sheet) and β-thigh DCLHE (SEQ ID NO:303) [the residue 308-312 of SEQ ID NO:38], stable β-interlayer that YGGLN (SEQ IDNO:304) [the residue 314-318 of SEQ ID NO:36] and KAIGN (SEQ ID NO:305) [the residue 330-334 of SEQID NO:36] (top beta sheet) are defined.This group beta sheet comprises, is arranged in the amino terminal and the carboxyl terminal of a stable secondary structure assembly.These truncates produce a kind of structure of functional domain closely according to believing.

Lee B//40HA1-2 construct (SEQ ID NO:39)For this construct, be by before the cysteine of the position 69 of SEQ IDNO:36, making the aminoterminal truncate, and behind the cysteine of position 289, make the c-terminus truncate, and keep four (4) individual intrinsic disulfide bond.Use simulation architecture and MMBD to check secondary structure, the amino terminal truncate is to be to cause in the ring that connects two groups of difference beta sheets in the amino terminal truncate.The amino terminal truncate is that 60 lysine place causes in the position, so that the residue 62-66[TQTRG that comprises SEQ ID NO:36 of film-long-range β-thigh group, SEQ ID NO:306] β-thigh still keep complete fully, and film-near-end β-thigh [TTTP of formation HA1-1 part, SEQ ID NO:300, the residue 48-51 of SEQ ID NO:36] eliminated fully.The carboxyl terminal truncate is that the serine in the position 300 of SEQ ID NO:36 causes.Truncate in this position keeps so that film-long-range β-thigh [KVIKG, SEQ ID NO:307, the residue 295-299 of SEQ ID NO:36] still all complete, and the secondary structure that film-near-end β-thigh [DCLHE, SEQ IDNO:308, the residue 308-312 of SEQ ID NO:36] is eliminated.Comprise film-long-range beta sheet of β-thigh TQTRG (SEQ ID NO:306) [the residue 62-66 of SEQ ID NO:36], SILHEV (SEQ ID NO:309) [the residue 97-102 of SEQ ID NO:36] and KVIKG (SEQ ID NO:307) [the residue 295-299 of SEQ ID NO:36], but be to be provided as it according to believing that conclusion becomes the stabilization secondary structure of tight functional domain structure.

B/ Malaysia/2506/2004, B/ Ohio/1/2005 and B/ Shanghai/361/2002 are according to same reference structure as the aforementioned.

Embodiment 3: clone and the performance of recombinant type dynein-hemagglutinin fused protein in escherichia coli

Materials and methods

The clone of influenza AHA subunitIndependent clone and express and derive from the HA ball head subunit of several A type influenza virus strains, or be fusant with flagellin.We are also with two kinds of protein that have been widely used in the conjugation vaccine as carrier protein, with HA ball head functional domain amalgamation and expression.CRM-197 be derive from diphtheria corynebacterium through sudden change diphtheria toxin, diphtherotoxin (DTx), and LTB is the B subunit of escherichia coli thermal instability enterogenous endotoxin.

These constructs are with wherein a kind of generation of following four kinds of methods:

Method #1: in this motion, be the fusion gene that will comprise flagellin (STF2) (SEQ ID NO:212) and HA subunit, carry out the codon optimization and be used for escherichia coli expression, and by chemosynthesis from commercial suppliers (DNA2.0Inc., cover Luo Gongyuan, CA) buy.Gene is gone out with NdeI and BlpI enzyme action, will insert fragment through gel-purified and be engaged to, with NdeI and BlpI digestion and the pET24a that handles with bacterial alkaline phosphatase (BAP) (Nova gold (Novagen), Santiago, CA).

Method #2: be easy clone and the gene that flagellin merges, satisfy generation and contain the card casket plasmid that the unique BlpI that is positioned at 3 of flagellin (STF2) gene (SEQ ID NO:212) ' end cuts the position.This is by importing silent mutation in nucleotide 5 '-GTGCTGAGCCTGTTACGT-3 ' of STF2 (SEQ ID NO:310) [nt1501 to 1518 of SEQ IDNO:212], produces unique BlpI and cut the position and finish in plasmid card casket pET24/STF2.blp (SEQ IDNO:51).To carry out the codon optimization for the antigenic synthetic gene of each target and be used for escherichia coli expression, and (DNA2.0Inc. covers Luo Gongyuan, CA) buys from commercial suppliers.Synthetic gene is gone out with the BlpI enzyme action, and via can compatible termination being bonded to, the pET24/STF2.blp that handles with BlpI and BAP.

Method #3: use for each construct specified forward and reverse primer (Keck FoundationBRLK, Yale University, Niu Hafen, CT; Authenticate reagent company (Midland Certified ReagentCo.), interior ground TX), is used in the dna profiling that is listed in each table and carries out pcr amplification interiorly.The PCR product is carried out BlpI digestion, through gel-purified and be engaged to, pET24/STF2.blp (the SEQ ID NO:51) carrier that gets by BlpI digestion and BAP Processing of Preparation in advance.

Method #4: by import flexible seven Yuans peptide connexons, Ser-Gly-Ser-Gly-Ser-Gly-Ser (S-G-S-G-S-G-S (SEQ ID NO:311)), and generation plasmid card casket pET24/STF2.SG (SEQ ID NO:52) in 3 of STF2 gene ' end.In the peptide connexon, produce a unique BlpI and cut the position, to help the HA subunit fragment of clone with the flagellin fusion.The synthetic gene that is used for escherichia coli expression through the codon optimization, be from commercial suppliers (DNA2.0Inc., cover Luo Gongyuan, CA) buy, it is cut out from maternal plasmid with BamHI and BlpI restriction endonuclease, and via can compatible termination being bonded to, the pET24/STF2 card casket of handling with BlpI and BAP.

In case out of the ordinary, the constructed plasmid that gets is to be used to transform competent escherichia coli TOP10 cell, and makes the recombinant that Analysis and Identification is inferred by PCR screening and estriction map.By the integrity of DNA sequencing proof construct, and use it for the conversion expressive host, BLR3 (DE3) (Nova gold, Santiago, CA; Cat#69053).Transformant is to screen in the flat board that contains kanamycin (50 μ g/mL), tetracycline (5 μ g/mL) and glucose (0.5%).Picking colony and inoculation are gone in the additional LB culture medium with 25 μ g/ml kanamycin, 12.5 μ g/ml tetracyclines and 0.5% glucose of 2ml, and grow overnight.These cultures of five equilibrium are used to inoculate the fresh culture of allocating with same medium, it is cultivated up to OD ₆₀₀=0.6, pass through to add 1mM IPTG this moment, and cultivated 3 hours and the induced protein expression down in 37 ℃.Collecting cell and analysing protein expresses.

SDS-PAGE and western blotting: by gel electrophoresis art and immunoblotting assay, measure protein expression and homogeneity.Cell is collected via centrifugal, and in the Laemmli buffer, dissolved.Each lysate of 10 μ l five equilibriums is diluted in, contains or do not contain in the SDS-PAGE sample buffer of 100mM DTT as Reducing agent.Sample is boiled 5 minutes, and carry out electrophoresis (SDS-PAGE) on application of sample to 10% sds page.With gel with the blue R-250 (Bio-Rad of Kao Maxi; Mo Lisi, CA) dyeing is to present protein band.For Western blotting, be that the capable cell lysates of 0.5 μ l/ is carried out electrophoresis, and through electrotransfer to pvdf membrane, blockade with 5% (w/v) milk powder again.

Then with film with anti--flagellin antibody (Inotek; Bei Fuli, MA), or influenza APR/8/38 convalescent period immune serum is surveyed.The PR/8/34 immune serum be in, the inferior deadly booster dose of accepting to get through measuring is 8 x 10 ¹The BALB/c mouse of the PR/8/34 influenza virus of the infectious dosage of egg (Jackson's laboratory, Ba Ergang, ME) the middle generation.Make animal after infection, recover then〉21 days, separate immune serum and clarificationization this moment.Treat that (IL) after the detection, (Pu Meijia (Promega), horse ground is gloomy, WI) presents protein band with alkali phosphatase colour generation substrate for Pierre Si, Luo Kelan with alkali phosphatase-conjugated secondary antibodies.Filter out to produce and have correct molecular weight, and with the pure strain of antibacterial of the protein band of suitable antibody response, be used to make protein for being used for bioanalysis.

By the synthetic gene approach described in method #1 and method #4, make derived from the HA of A/ Puerto Rico/8/34 strain (PR8) (SEQ ID NO:1) and be shown in the construct of table 1.Equally, construct derived from A/ Vietnam/1203/2004 strain (SEQ ID NO:2) is shown in table 2, construct derived from the HA of A/ Indonesia/2005 strains (IND) (SEQ ID NO:3) is shown in table 3, and the construct that derives from A/ New Caledonia/12/99 strain (NC) (SEQ ID NO:4) is shown in table 4." IND " is used for this paper and means " Indonesia ".The dna primer and the dna profiling that are used for pcr amplification reaction are shown in same table if suitably.

Table 1: the PR8HA construct that is used for expression in escherichia coli

SEQ ID NO：	Construct	Method
			53	STF2.HA1-1	#4
54	STF2.HA1-1.his	#4
			55	STF2.HA1-2	#1
56	STF2.HA1-2mut	#				1
			57	STF2.HA1-3	#1
58	STF2.HA1-3mut	#				1
			59	HA1-1	#1
60	HA1-1.his	#1
			61	HA1-2.his	#1
62	CRM.HA1-2	#1
			63	LTB.HA1-2	#1

Table 2: the VN HA construct that is used for expression in escherichia coli

SEQID NO：	Construct	Method	The SEQ ID NO of primer:	REV primer SEQ ID NO:	Dna profiling SEQ ID NO:
						64	STF2.HA1-1	#3	65	66	67
68	STF2.HA1-2	#3	69	70	67
						71	STF2.HA1-2mut	#2	N/A	N/A	72

Table 3: the IND HA construct that is used for expression in escherichia coli

SEQID NO：	Construct	Method	The SEQ IDNO of primer:	REV primer SEQ ID NO:	Dna profiling SEQ ID NO:
						73	STF2.HA1-1	#3	74	66	75
76	STF2.HA1-2	#3	69	77	75
						78	STF2.HA1-2mut	#2	N/A	N/A	79

Table 4: the NC HA construct that is used for expression in escherichia coli

SEQID NO：	Construct	Method	The SEQ IDNO of primer:	REV primer SEQ IDNO:	Dna profiling SEQ IDNO:
						80	STF2.HA1-1	#3	81	82	83
84	STF2.HA1-2	#3	85	86	83
						87	STF2.HA1-2mut	#2	N/A	N/A	88

The result

Protein carrier has been widely used in human vaccine.Cross reactivity material (the CRM of diphtheria toxin, diphtherotoxin ₁₉₇) be considered to, favourable is used for several conjugation vaccine concoctions as carrier molecule.Exemplary carrier comprises escherichia coli thermal instability enterogenous endotoxin (LT) and its B subunit (LTB), tetanus toxoid (TT) and vibrio cholera toxoid (CT).Use CRM ₁₉₇With the representative of LTB as this full carrier protein, we have produced the HA ball head of wherein A/ Puerto Rico/8/34 strain (PR8) (SEQ ID NO:1), be to merge to the construct of 3 ' end of CRM197 gene or LTB gene, and produce construct CRM.HA1-2 (SEQ ID NO:62) and LTB.HA1-2 (SEQ ID NO:63) with method #1.Described construct proves via the DNA sequencing, and is used to transform expressive host BLR3 (DE3) (Nova gold, Santiago, CA; Cat#69053).

Transformant is screened in the flat board that contains kanamycin (50 μ g/mL), tetracycline (5 μ g/mL) and glucose (0.5%).The cultivation overnight of several bacterium colonies of picking is used to inoculate the fresh LB culture medium of replenishing with 25 μ g/ml kanamycin, 12.5 μ g/ml tetracyclines and 0.5% glucose with culture.In OD ₆₀₀=0.6 o'clock, cultivate 3 hours induced proteins with 1mM IPTG down in 37 ℃ and express.Collect cell, and with the lysate of five equilibrium on 10% SDS-PAGE, by the blue dyeing of Kao Maxi, and analyze by the immunoblotting that uses the PR/8/34 convalescent serum.In the case of CRM.HA1-2 construct, be several bacterium colonies of picking and analyze express by SDS-PAGE.

When analyzing by the blue dyeing of Kao Maxi SDS-PAGE gel, pure again strain all presents and has obvious MW 84Kda, and the band consistent with expection MW.In matched group culture (not adding IPTG person), this band do not occur, represent that it is inductive by the IPTG specificity.This observes at the cell extraction thing with two pure strain #5 and #6, in having or not having under Reducing agent (5mM DTT) existence and carry out a grade timesharing, obtains further to confirm.Its disulfide bond is handled destructive recombinant protein by DTT, can be by relevant antibody recognition, but natural recombinant protein can.Similarly, the pure strain of expression construct LTB.HA1-2 presents the consistent band with the predicted molecular weight 39.8KDa of institute, when the protein with the IPTG abduction delivering.The homogeneity of LTB-HA1-2 fusion rotein is to confirm by the western blot analysis that uses the mice convalescent serum.When existing, its intensity subtracts this band as Reducing agent (beta-mercaptoethanol).As if this observation hint described later, the reduction dosage that this experiment is used is not enough.Comprehensive data support shown in this article, the HA ball head can successfully merge to carrier protein, and produces sensitive proteinic conception on the configuration.

Recombinant type dynein-the clone of hemagglutinin fused protein in escherichia coli

The clone of HA influenza B subunitFrom several Type B influenza virus strains clone the HA ball head, and performance is the fusant with flagellin.These constructs are to produce by two step PCR.

The HA subunit is used for escherichia coli expression through the codon optimization, and (DNA2.0Inc. covers Luo Gongyuan, CA) buys from commercial suppliers with chemosynthesis.Use synthetic DNA to carry out pcr amplification HAI-1 or HA1-2 as template.Flagellin (STF2) sequence (SEQ ID NO:212) is derived from plasmid pET24a-STF2.HA1-2.The STF2DNA fragment is carried out pcr amplification, and the C-of PCR product end and the N-terminal sequence that merges the HA subunit, have that one section 28-30bp's is overlapping.

STF2 and HA subunit are passed through 2 ^NdPCR merges.Use be shown in the forward out of the ordinary of following table and reverse primer (dna integration science and technology, Inc, Carlow paddy, IA52241), from the dna profiling that also is shown in following table increase fused protein DNA.Then the PCR product is carried out XbaI and decompose, gel-purified and be engaged to the pET24a-STF2.HA1-2 that digests with XbaI and SnaBI in advance.

Described constructive system is shown in following table, if suitably, also list the dna primer and the dna profiling that are used for pcr amplification reaction.

The FluB STF2.HA construct that is used for expression in escherichia coli

SEQID NO：	Construct	The SEQ ID NO of primer:	REV primer SEQ ID NO:	Dna profiling SEQ ID NO:
					184	STF2.HA1-1(MAL)	193，195	194，196	55，183
186	STF2.HA1-2(MAL)	193，198	197，199	55，185
					188	STF2.HA1-2(SH)	193，201	200，202	55，187
190	STF2.HA1-2(Lee)	193，204	203，205	55，189
					192	STF2.HA1-2(Ohio)	193，207	206，208	55，191

In case out of the ordinary, the constructed plasmid that gets is to be used to transform competent e.colidh5, and makes the recombinant that Analysis and Identification is inferred by estriction map.By the integrity of DNA sequencing proof construct, and use it for the conversion expressive host, BLR3 (DE3) (Nova gold, Santiago, CA; Cat#69053).Transformant is in the LB (Veggie peptone, EMD bioscience, La Jolla, the CA that contain kanamycin (35 μ g/mL); Cat#71280; Veggie yeast extract, EMD bioscience, Cat#71279; ) flat board screens.Picking colony and inoculation are gone in additional LB (Veggie) culture medium with 35 μ g/ml kanamycin of 2ml, and grow overnight.These cultures of five equilibrium are used to inoculate the fresh culture of allocating with same medium, it is cultivated up to OD ₆₀₀=0.6.Express by adding 1mM IPT G induced protein.After cultivating 2 hours under 37 ℃, collect cell and analyze its protein expression.

SDS-PAGE and western blotting: by gel electrophoresis art and immunoblotting assay, measure protein expression and homogeneity.Cell is collected via centrifugal, and (50mM Tris, 200mMNaCl pH8.0) dissolves in the buffer in Tris/NaCl.Each lysate of two five equilibriums is diluted in the SDS-PAGE sample buffer, and boiled 5 minutes.Will be wherein a five equilibrium sample with reduction DTT (final concentration 200mM, the EMD bioscience, La Jolla, CA 92039; Cat#233155).Sample (the capable cell lysates of 1.5 μ l/) application of sample is carried out electrophoresis (SDS-PAGE) to the 4-12% sds page.With gel with the blue R-250 (Bio-Rad of Kao Maxi; Mo Lisi, CA) dyeing is to present protein band.

For Western blotting, be that the repetition gel is carried out electrophoresis, and be transferred on the pvdf membrane through point, blockade with 5% (w/v) milk powder again.Then with film with anti--flagellin antibody (6H11, Inotek, Bei Fuli, MA; Lot#1030B5), or at B/ Malaysia (2506/2004, CDC#2005741987) the ferret antiserum of Chan Shenging is surveyed.Treat with mountain horseradish peroxidase (HRP)-conjugated secondary antibodies (1:10,000) (goat is anti--mice is available from Jackson's immune Research laboratory; Xi Geluofu, the PA. goat is anti--and ferret is available from the Bei Xier laboratory; The Montgomery, TX.) survey after, in to add HRP colour generation substrate (Pierre Si; The Lip river Crane, IL; Cat#34018) present protein band.About molecular weight of STF2.HA1-1 (MAL) is 84kDa; And about molecular weight of STF2.HA1-2 (MAL and SH) is～78kDa.The microorganism of application of sample two STF2.4 * M2e fusion rotein, as the positive controls of anti--flagellin antibody, and the negative control group of anti--HA serum.Comprise that in this experiment (B/ Malaysia 2506/2004 CDC#2005756250), as the positive controls of anti--HA serum, reaches the negative control group of anti--flagellin antibody to the virolysis product.The blue dyeing of the Kao Maxi of gel shows, low STF2.HA1-1 (MAL) protein of inducing; But STF2.HA1-2 (MAL) and the proteic band of STF2.HA1-2 (SH) are a lot of by force.Use the western blot analysis of anti--flagellin monoclonal antibody (1:4000), present the strong positive band of about 84kDa at the row of application of sample STF2.HA1-1 (MAL), and present about 75kDa band in the row of application of sample STF2.HA1-2 (MAL) or STF2.HA1-2 (SH).Each fusion rotein is in the pattern of inducing of Western blotting, with very similar through the result of the blue dyeing of Kao Maxi gel.Without reduction and through going back crude protein, resisted-degree of flagellin antibody recognition is suitable.It is anti--flagellin that (positive controls of STF2.4 * M2e) shows single strong band, and negative control group does not then have and presents any band.

Use the western blot analysis of anti--HA polyclonal antiserum (1:1000), present and the similar pattern of trace with anti--flagellin, only through reductive albumen compared to without its signal weakening of reduction sample, expression exists the major part in this antiserum to resist-the HA polyclonal antibody, be that identification forms formed configuration epitope by correct disulfide bond, and the fraction antibody in the antiserum, identification does not rely on the linear epitope that disulfide bond forms.Use is caught elisa assay and is observed analog result.Notice valuably, also discern, B/ Shanghai/361/2002 that belongs to the chevron kind and be at the antiserum of B/ Malaysia/2506/2004 (Victoria plant be).This result points out that these two kinds is not only total many primary sequence similaritys, also total tertiary structure.

Western blotting and ELISA result determine that functional influenza B HA1-1 of tool and HA1-2 protein expression are in escherichia coli.

Embodiment 4: the purification and the biochemical characteristicsization of recombinant type dynein-hemagglutinin fused protein

Materials and methods

Bacterial growth and cytolysisThe HA construct is expressed among the host strain BLR (DE3).To have the Bacillus coli cells of the plasmid of coding STF2.HA1-1.his (PR8) (SEQ ID NO:54), and cultivate as previously mentioned and gather in the crops.Bacterium out of the ordinary is recovered from the glycerol reserve, and grow in shake in the bottle to final volume be 12L.Cell is grown in the LB culture medium that contains 25 μ g/ml kanamycin/12.5 μ g/ml tetracycline/0.5% glucoses to OD ₆₀₀=0.6, and cultivate down in 37 ℃ with 1mM IPTG and to induce in 3 hours.Cell is collected by centrifugal (7000rpm x 7 minutes in Sorvall RC5C centrifuge), and resuspending is in 1X PBS, 1% glycerol, 1 μ g/ml DNAseI, 1mM PMSF, protease inhibitor cocktail and 1mg/ml lysozyme.Then with cell in 15, the dissolving that twice makes by little liquefier under the 000psi.With lysate in Beckman (Beckman) Optima L supercentrifuge (Beckman colter (Beckman Coulter); Fu Ledun, CA) in, in 45, under the 000g centrifugal one hour so that solubility is partly separated with insolubility.

From escherichia coli purification STF2.HA1-1.his (PR8) (SEQ ID NO:89) protein and folding againThrough dissolving with centrifugal after, in 100ml1X TBS, in the pH8.0+1mM beta-mercaptoethanol, and solid urea is added to final concentration is 6.2M with insolubility (precipitation) part resuspending.Treat to carry out mixture centrifugal as described above with behind the Dounce glass bead homogenizer resuspending.With the supernatant application of sample of gained to filling with NiSO ₄, and in the equilibrated chelating agarose tubing string of buffer A [20mM Tris, pH 8.0/8M carbamide/0.5M NaCl] (GE/ Ai Moxun (GE/Amersham Biotechnology) bioscience; Skin SIKA tower prestige, NJ) on.

Treat with after 1L buffer B [buffer A+1% (w/v) TX-100] flushing, with tubing string in 5-tubing string volume from 100% buffer A to 100% buffer C[buffer A+0.5M imidazoles] linear gradient carry out eluting.Compile peak stream part, in Amicon 15 centrifugal concentrators (Mi Libo (Millipore); The Baily card MA) concentrates 3.5 times, and to 2 liters of 8M carbamide/20mM Tris of 3 x, pH 8.0/2mM EDTA dialysis.After dialysis, is that 0.1mg/ml protein is in folding buffer [0.1M Tris again with STF2.HA1-1.his (PR8)-(SEQ ID NO:89) by rapid dilution to final concentration, pH 8.0/0.1M NaCl/1% (w/v) glycerol/5mM reduced glutathion/1mM oxidized form of glutathione] in, and cultivation is spent the night and is carried out folding again under room temperature.

To fill with NiSO in 5ml through folding again protein capture then ₄, and in buffer D[20mMTris, pH 8.0/0.5M NaCl/1mM reduced glutathion/0.2mM oxidized form of glutathione] equilibrated chelating agarose HiTrap tubing string (GE/ Ai Moxun bioscience; Skin SIKA tower prestige, NJ) on, and 100% buffer E[buffer D+0.5M imidazoles] in carry out eluting.Compile peak stream part, use the centrifugal concentrator (Mi Libo of Amicon15; The Baily card MA) concentrates, and in through 1X TBS, the equilibrated Superdex 200 size exclusion tubing strings of pH 8.0+0.5% (w/v) deoxycholic acid Na (GE/ Ai Moxun bioscience; Skin SIKA tower prestige NJ) is carried out fraction.Compile peak stream part, to 3 x 2L of 1X TBS, pH 8.0 (not containing dexycholate) dialysis is bottled and is stored in-80 ℃.

STF2.HA1-2 (PR8) (SEQ ID NO:90) and STF2.HA1-2mut (PR8) (SEQ ID NO: 91) purification: will have the Bacillus coli cells of the plasmid of coding STF2.HA1-2.his (PR8) (SEQ ID NO:55), and cultivate as previously mentioned and gather in the crops.Through dissolving with centrifugal after, will contain insolubility (precipitation) the part resuspending of STF2.HA1-2 (PR8), and use Dounce homogenizer homogenizes among pH 8.0+0.5% (w/v) the Triton X-100 in 50mM Tris.Then with homogenizing fluid centrifugal (19,000rpm x 10min).Then with the precipitation part of gained in 50mM Tris, homogenize again among pH 8.0+0.5% (w/v) the Triton X-100+1.0M NaCl, and centrifugal.Then inclusion body part (precipitation) is dissolved in the buffer A [50mM acetic acid Na, pH4.0+8.0M carbamide], and centrifugal (19,000rpm x 10min.) are to remove the insolubility fragment.

With the supernatant of gained in equilibrated sucrose S tubing string (GE/ Ai Moxun bioscience in buffer A; Skin SIKA tower prestige NJ) is carried out fraction, and in 5 tubing strings-volume with the linear gradient of buffer B (50mM acetic acid Na, pH4.0,1.0M NaCl) in eluting.Then protein is carried out folding to buffer C (100mM Tris-HCl, pH 8.0) by rapid dilution again.Then will be through folding protein in equilibrated Q agarose HP tubing string (GE/ Ai Moxun bioscience in buffer C; Skin SIKA tower prestige is carried out fraction on NJ), and in 0% to 60% linear gradient of buffer D (buffer C+1MNaCl) eluting.Then with Q HP eluate in through 1X Tris-buffering saline solution, pH 8.0 equilibrated Superdex 200 size exclusion chromatographic analysis (SEC) tubing string (GE/ Ai Moxun bioscience; Skin SIKA tower prestige NJ) is carried out fraction, with monomer and accumulative Separation of Proteins.Compile monomer flow part then, five equilibrium also stores in-80 ℃.

The purification of STF2.HA1-3 (PR8) (SEQ ID NO:92): will have the Bacillus coli cells of the plasmid of coding STF2.HA1-3 (PR8) (SEQ ID NO:57), and cultivate as previously mentioned and gather in the crops.Through dissolving with centrifugal after, solubility (supernatant) part that will contain STF2.HA1-3 (PR8) is adjusted to 6.2M carbamide, 100mM DTT also cultivates to spend the night under room temperature and also protein is reduced and degeneration fully.Then five times in protein is diluted in the buffer A (50mM citric acid, pH 3.5,8M carbamide, 1mM beta-mercaptoethanol, 1mM EDTA), and application of sample is extremely with the equilibrated SP agarose of buffer A FF tubing string (GE/ Ai Moxun bioscience; Skin SIKA tower prestige, NJ) on.Be to remove endotoxin, satisfy tubing string, wash with 10 tubing string volume buffer C (50mM citric acid, pH 3.5,1mM beta-mercaptoethanol, 1mM EDTA, 70% (v/v) isopropyl alcohol) more subsequently with buffer B (buffer A+1% (w/v) Triton X-100).

Then with conjugated protein in five tubing string volume buffer A: eluting in the linear gradient of buffer D (buffer A+1M NaCl).The protein that eluting is gone out is dialysed to buffer E (20mM Tris, pH 8.5,8M carbamide, 1mM beta-mercaptoethanol, 1mM EDTA) then, and by sucrose Q tubing string (GE/ Ai Moxun bioscience; Skin SIKA tower prestige, NJ).In circulation stream part, collect STF2.HA1-3 (PR8) (SEQ ID NO:92) protein.Protein is used Amicon 15 centrifugal concentrator (Mi Libo; The Baily card MA) concentrates, and buffer F (20mM Tris, pH 8.0,8M carbamide, 1mM EDTA) is dialysed to remove Reducing agent.To be 0.1mg/ml by rapid dilution to final concentration through the polypeptide of degeneration then, in folding buffer [0.1M Tris again, pH 8.0/0.1M NaCl/1% (w/v) glycerol/5mM reduced glutathion/1mM oxidized form of glutathione] in carry out again foldingly, and under room temperature, cultivate and spend the night.Then will be through folding again protein in cushioning saline solution (TBS) through 1X Tris-, pH 8.0, add on the equilibrated Superdex200 size exclusion of 0.25% (w/v) the sodium cholate tubing string and carry out fraction, with monomer STF2.HA1-3 (PR8) and accumulative Separation of Proteins.Compile monomer flow part, to remove cholate, five equilibrium also stores in-80 ℃ to dialysis.

The purification of HA1-2His (PR8) (SEQ ID NO:93): will have the Bacillus coli cells of the plasmid of coding STF2.HA1-2.his (PR8) (SEQ ID NO:61), and cultivate as previously mentioned and gather in the crops.Through the dissolving with centrifugal after, insolubility is partly homogenized, and with buffer A (50mM Tris, pH 8.0) cleaning once, clean with buffer+0.5% (w/v) Triton X-100 and buffer A+0.5% (w/v) TritonX-100+1M NaCl in regular turn subsequently.Then the insolubility material is cleaned twice with independent buffer A again.Solubilization of inclusion bodies that then will be through cleaning is in buffer B (1X PBS, pH 7.4,8M carbamide), and application of sample is to filling with NiSO ₄Ni-NTA (sigma-Ya capable strange (Sigma-Aldrich); The Saint Louis city, MO) tubing string, and in the linear gradient of buffer C (50mM Tris, pH 8.0,8M carbamide, 0.3M NaCl, 0.5M imidazoles) eluting.Be to remove endotoxin, satisfy protein through eluting to dialysed overnight, and application of sample is to the Ni-NTA tubing string.

Then tubing string is washed with buffer D (10mM Tris, pH8.8,8M carbamide, 60% (v/v) isopropyl alcohol), and carry out eluting as described above.To be diluted to by 1:0 (v/v) through the protein of Ni-NTA purification then again in the folding buffer (50mM Tris, pH 8.8) and carry out folding again.To use Amicon ultra-filtration unit (Mi Libo through folding again HA1-2His (PR8) (SEQ ID NO:93) protein then; The Baily card MA) concentrates, and application of sample is to through 1X TBS, pH 8.0 equilibrated Superdex 200 size exclusion tubing strings (GE/ Ai Moxun bioscience; Skin SIKA tower prestige NJ) is carried out fraction.Compile monomer flow part, five equilibrium also stores in-80 ℃.

SDS-PAGE and western blotting: measure the homogeneity of all HA constructs by SDS-PAGE, and assess purity.The sample of 5 μ g five equilibriums is diluted in, contains or do not contain in the SDS-PAGE sample buffer of 100mM DTT as Reducing agent.Sample is boiled 5 minutes, and application of sample to 10% sds page (LifeGels; The blue Si Senlin of method, Xin Nanweiersi, AUS) on, and carry out the electrophoresis art.With gel with the blue R-250 (Bio-Rad of Kao Maxi; Hull Ke Lishi, CA) dyeing is to present protein band.For Western blotting, be that the capable overall protein matter of 0.5 μ l/ is carried out electrophoresis as previously mentioned, through electricity-be transferred on the pvdf membrane, and blockade, then afterwards with anti--flagellin antibody (Inotek with 5% (w/v) milk powder; Bei Fuli, MA), or influenza A PR/8/38 convalescent period immune serum (following in Proteantigen ELISADescribed) survey.Treat that (IL) after the detection, (Pu Meijia, horse ground is gloomy, WI) presents protein band with alkali phosphatase colour generation substrate for Pierre Si, Luo Kelan with alkali phosphatase-conjugated secondary antibodies.

Protein analysis: use Micro BCA (two cinchoninic acids) to analyze (Pierre Si, Luo Kelan IL), use bovine serum albumin,, measure the overall protein matter concentration of all proteins according to the explanation of manufacturer as reference material with the micro plate form.

Endotoxin analysis: for the endotoxin concns of all proteins, be to use the QCL-1000 LAL test kit that quantitatively develops the color, the explanation that is used for the micro plate method according to manufacturer is measured.

The TLR5 biological activity is analyzed: the HEK293 cell is expressed TLR5 inherently, and in the reaction to the TLR5 citation, secretes several soluble factors (comprising IL-8).With cell inoculation (50,000 cells/well) in the micro plate of 96-hole, and following test protein added and cultivate and spend the night: STF2.HA1-1His (PR8) (SEQ ID NO:89); STF2.HA1-2 (PR8) (SEQ ID NO:90); STF2.HA1-2 (PR8) mut (SEQ ID NO:91); STF2.HA1-3 (PR8) SEQ ID NO:92); HA1-2His (PR8) (SEQ IDNO:93); STF2.HA1-2 (Mal) (SEQ ID NO:211) supernatant; STF2.HA1-2 (Mal) (SEQID NO:211) is through folding again; STF2.HA1-2 (SH) (SEQ ID NO:211) supernatant; STF2.HA1-2 (SH) (SEQ ID NO:211) is through folding again; STF2.HA1-1 (Mal) (SEQ ID NO:209) supernatant; And STF2.HA1-1 (Mal) (SEQ ID NO:209).Next day, collect conditioned medium, be transferred to clean 96-hole micro plate, and freezing down in-20 ℃.After waiting to thaw, in the antibody that uses Anti-Human's class-IL-8 to cooperate to (Pierre Si, Luo Kelan, IL; #M801E and#M802B) sandwich ELISA exists according to the IL-8 in the explanation analysis condition culture medium of manufacturer.Use micro plate spectrophotometer (FARCyte, GE/ Ai Moxun bioscience; Skin SIKA tower prestige NJ) is measured optical density (OD).

Proteantigen ELISA: whether correctly show for measuring recombination fusion protein, satisfy the antigenicity of assessing HA-fusion rotein out of the ordinary by ELISA through folding HA epitope.96-hole elisa plate with serial dilution each target protein (starting from 5 μ g/ml) in PBS (100 μ l/ hole), is applied under 4 ℃ and spends the night.With the analysis dilution buffer liquid (ADB of plate with 200 μ l/ holes; BD Fa Minggen), under room temperature, blockaded one hour, in PBS-T, clean three times then.Then the one-level antibody with fixed dosage adds to each hole.

For analyzing the HA reactivity, to satisfy 100 μ l/ hole 1:0,000 non-immunity or the PR/8/34 convalescent period immune serum that is diluted in ADB adds.The PR/8/34 immune serum is that the inferior deadly booster dose that gets through measuring in acceptance is 8x10 ¹The BALB/c mouse of the PR/8/34 influenza virus of the infectious dosage of egg (Jackson's laboratory, Ba Ergang, ME) the middle generation.Make animal after infection, recover then〉21 days, separate immune serum and clarificationization this moment.For flagellin or the histidine-tagged ELISA of 6x, be (because of Vito gold (invitrogen) with anti-6x His; Ka Sibeide, CA), or flagellin (Inotek; Bei Fuli, MA) monoclonal antibody is dissolved in ADB (100 μ l/ hole) adding with 1 μ g/ml, and plate was cultivated under room temperature 1 hour, or spends the night under 4 ℃.Then plate is cleaned three times with PBS-T.The HRP-labelling goat that is diluted in ADB is resisted-mouse IgG antibody (Jackson's immunochemistry (Jackson Immunochemical); Xi Geluofu PA) adds (100 μ l/ hole), and plate was cultivated under room temperature 1 hour.Plate is cleaned three times with PBS-T.TMB Ultra substrate to be added (Pierre Si, Luo Kefu, IL), and behind the monitoring color colour generation, micro plate spectrophotometer (FARCyte, GE/ Ai Moxun bioscience; Skin SIKA tower prestige NJ) is measured A ₄₅₀

Result and discussion

Protein output and purity: the reorganization HA that makes in escherichia coli and the purification result of STF2.HA fusion rotein are shown in the table 6.All four kinds of protein all make with high yield, have the purity of estimation and surpass 90%, and endotoxin quite is lower than reference material acceptable concentration 0.1EU/ μ g.Three kinds of STF2 fusion rotein also have the very high biological activity of TLR5 in vitro, and HA1-2His ₆(SEQ ID NO:93) protein (as expected) does not have the TLR5 activity.

Table 6

Protein	SEQID NO：	Output (mg)	Purity est. (%)	Endotoxin (EU/ μ g)	TLR5 activity (EC 50，ng/ml)
						STF2.HA1-1His(PR8)	89	6.4	>90	0.04	0.2
STF2.HA1-2(PR8)	90	6.0	>98	0.02	0.15
						STF2.HA1-2(PR8)mut	91	12	>95	0.01	1.2
STF2.HA1-3(PR8)	92	2.1	>90	0.016	1.2
						HA1-2His(PR8)	93	10	>95	0.03	nd

The proteic antigenicity of influenza AHA that makes in escherichia coli: four kinds of recombiant proteins are passed through to resist the antibody (Inotek of STF2 (flagellin); Bei Fuli MA), and collects the western blotting that the immune antiserum from PR/8/34 convalescent period mice carries out and analyzes.As if three kinds of STF2 fusion rotein work as with the reacting phase of anti--flagellin antibody.STF2.HA1-1His (PR8) (SEQ ID NO:89), STF2.HA1-2 (PR8) (SEQ ID NO:90) and HA1-2His (PR8) (SEQ ID NO:93) also react with anti--PR/8/34 convalescent serum.This reactivity also is detected in without reductive protein sample; DTT is added to sample buffer lower this signal widely.This finds hint, and most of resisting-HA antibody is dependency on the configuration in the convalescent serum, and the suitable disulfide bond knot in HA, is essential for the reactivity by Western blotting.With respect to these results, STF2.HA1-3 (PR8) (SEQ ID NO:92) does not react with convalescent serum effectively, and the weak signal that is produced is not influenced by Reducing agent.This finds hint, and STF2.HA1-3 protein may not suitably fold, and/maybe can not represent natural HA epitope.

Be used for the proteic ELISA of catching of influenza B ball head

The antigenicity that ELISA assesses HA-fusion rotein out of the ordinary is caught in use.Anti--the flagellin monoclonal antibody is to be used for these albumen of solid-phase capture.Be used in the ferret that produces when infect natural B/Malaysia/2506/2004 and resist-serum, detect the HA-fused protein of being caught.The result of this analysis will determine whether recombination fusion protein is showed through correct folding HA epitope.Have rational sequence homology owing to different influenza B ball head are intermolecular, expection 84.6% these antiserums can react with Malaysia and Shanghai ball head construct.

The preparation of sample

The escherichia coli that to express STF2.HA1-2 (Malaysia (SEQ ID NO:210)), STF2.HA1-2 (Shanghai (SEQID NO:211)) and STF2.HA1-1 (Malaysia (SEQ ID NO:209)) recombiant protein are by centrifugation, and resuspending is in Tris buffer (50mM Tris, 200mM NaCl, pH 8) in.With cell with the shatter dissolving of ultrasound.Cell lysates is centrifugal, so that soluble protein and insolubility Separation of Proteins.With insolubility protein precipitation resuspending in the Tris buffer that contains 6M carbamide, and with protein by with this solution rapid dilution 1:0 doubly to the Tris buffer, and carry out folding more fast.With UV ₂₈₀(spectrophotometer DU 800) comments the meter soluble protein, with the protein concentration of the precipitation sample that contains again unfolded protein.Contain STF2.HA1-1 (Mal) (SEQ ID NO:209) by the ELISA assessment; STF2.HA1-2 (Mal) (SEQ ID NO:210), STF2.HA1-2 (SH, Shanghai) (SEQ ID NO:211) proteinic supernatant, and contain through folding the dissolution precipitation of STF2.HA1-1 (Mal), STF2.HA1-2 (Mal), STF2.HA1-2 (SH) again, with the sero-fast reactivity of ferret of generation when natural at B/ Malaysia/2506/2004 infection.Use escherichia coli supernatant sample, and it is proteinic through dissolving, folding sample is as negative control group again to contain STF2.4xM2e (SEQ ID NO:94).

The ELISA method

Is that (Inotek, Bei Fuli MA), and cultivate down in 2-5 ℃ and to spend the night for the flagellin monoclonal antibody specific 6H11 of 0.5ug/mL with elisa plate (Maxisorp, Nuo Ke, Denmark) coated with concentration.The solution that will contain antibody is drained, and with 300uL/ hole Super Block+Tween-20, blockades under 25 ℃ 2 hours.Flat board is cleaned once and pats dry with 1xPBS.Carry out three times of serial dilutions of different proteins solution in the dilution plate, initial concentration is 5ug/mL.100uL is transferred to elisa plate from the dilution plate.

Plate was cultivated 1 hour down in 25 ℃.By plate is cleaned 3 times with PBS+0.05%Tween-20, and with unconjugated protein removal.To be diluted to 1:00 at the ferret antiserum that B/ Malaysia/2506/2004 (CDC, AL, Georgia) produces, and 100uL will be added to each hole.Then plate was cultivated 1 hour down in 25 ℃.After this incubation step, flat board is cleaned 6 times with the PBS that contains 0.05%Tween-20.Will through conjugation to the mountain horseradish peroxidase (goat IL) is anti-for HRP, Bei Xier laboratory Inc. (Bethyl Labs Inc.)-ferret IgG dilutes 1:0,000, and 100uL added to each hole.Then plate was cultivated 30 minutes.After this incubation step, flat board is cleaned 6 times with PBS+0.05%Tween-20.100uL is contained HRP substrate 3,3 ', 5, and (Pierre Si, Luo Kefu IL) add to each hole to the TMB Ultra of 5 '-tetramethyl benzidine.Behind this substrate to be added, monitoring color colour generation, and to add 100uL/ hole 1M H ₂SO ₄Cessation reaction.(SpectraMax 190, molecular device (Molecular devices), sun paddy, CA) the last A that measures to read machine in the micro plate meter ₄₅₀

The proteic antigenicity of influenza B HA that makes in escherichia coli

ELISA data (Fig. 3) point out that antiserum can be discerned the epitope to the Reducing agent sensitivity.Protein is handled with Reducing agent, and it changes protein configuration by destroying disulfide bond, and lowers antiserum and described proteinic reactivity.The overall lysate of STF2.HA1-2 (Mal) without reduction, the overall lysate of STF2.HA1-2 (SH) without reduction and the overall lysate of STF2.HA1-1 (Mal) without reduction, when remaining in suitably folding configuration, its reactivity is very good, the configuration of representing HA-fusion rotein out of the ordinary is suitable with the proteic ball head configuration of natural HA.Though antiserum contains the antibody to the configuration sensitivity, and reactive rely on correct disulfide bond, it also contains the insensitive antibody of disulfide bond, and this is by under reduction and non-reduced condition, all observes residual activity and obtains proof.

The proteic TLR5 biological activity of STF2.HA (influenza B)

Recombination fusion protein presents with deriving from catches the effect that ELISA result conforms to, and expression has more activity (Fig. 4 and 5) through folding sample than the lysate of native form.The sample that is natural unprocessed form shows that it is without the few 2-of folding sample activity doubly.These protein are false folding in native form, and therefore need folding step to recover the TLR5 biological activity again.This is active active consistent with the HA in catching ELISA.

Embodiment 5: represent the immunogenicity of Strain A/ Puerto Rico/8/34 in colibacillary recombinant type dynein-hemagglutinin fused protein

Materials and methods

Zooscopy: use the 6-8 female BALB/c mouse in age in week (Jackson's laboratory, Ba Ergang, ME).With 10 every group of mice group, and near accepting groin in the 0th and 14 day subcutaneous (s.c) cause exempt from as follows:

1) PBS (phosphate-buffered saline solution)

2) STF2.4xM2e of 3 μ g (SEQ ID NO:94) is in saline solution buffer (10mM histidine, 10mM Tris, 75mM NaCl, 5% (vol/vol) sucrose, 0.02% (w/v) gathers sorbose acid esters-80,0.1mM EDTA, 0.5% (v/v) ethanol, pH7.2)

3) STF2.HA1-2 of 30 μ g (PR8) (SEQ ID NO:90) is in PBS

4) STF2.HA1-2 of 3.0 μ g (PR8) (SEQ ID NO:90) is in PBS

5) STF2.HA1-2 of 0.3 μ g (PR8) (SEQ ID NO:90) is in PBS

6) STF2.HA1-2 of 0.03 μ g (PR8) (SEQ ID NO:90) is in PBS

Extra one group of five mice is accepted to excite with 8x10 through inferior the causing death that measuring gets ¹Infectious dosage (EID) PR/8/34 of egg, and make its recovery〉21 days.Then with these animals during exciting research (referring to following), use as immune restoration phase positive controls.With mice (the short liter) blood sampling in the 10th day (elementary) and 21 days, and with serum by condensing and centrifugal clarificationization, and store under-20 ℃.

Serum antibody is measured: measure HA-specific IgG concentration by ELISA.(Costar (Cat#9018) Corning, NY) under 4 ℃, (making in fruit bat) (SEQ ID NO:176) is dissolved in PBS (5 μ g/ml) and applies and spend the night with 100 μ l/ hole HA0sHis protein with 96-hole elisa plate.With the analysis dilution buffer liquid (ADB of plate with 200 μ l/ holes; BD Fa Minggen, (Cat#:555213) (famous scenic spot tooth brother CA) blockaded under room temperature one hour.Plate is cleaned three times in PBS+0.05% (v/v) Tween 20 (PBS-T).Serum is added (100 μ l/ hole) in the diluent of ADB, and plate cultivation under 4 ℃ is spent the night.Plate is cleaned three times with PBS-T.The HRP-labelled goat that is diluted in ADB is resisted-mouse IgG antibody (Jackson's immunochemistry; Xi Geluofu, PA (Cat#:115-035-146)) add (100 μ l/ hole), and plate was cultivated under room temperature 1 hour.Plate is cleaned three times with PBS-T.(IL), and behind the monitoring color colour generation, (Dole breathes out TMB Ultra substrate to be added, NC) measures A on the micro plate spectrophotometer in Tecan Farcyte for Pierre Si, Luo Kefu ₄₅₀

The full cell ELISA of MDCK: with mdck cell (ATCC (Cat#CCL-34) Manassas, VA) in the 96-well culture plate (BD (Cat 353075), healthy and free from worry, NY), in the DMEM complete medium that contains 10% FCS, in 37 ℃ down growth one to two day or up to cell near being paved with.Then with each hole and 1 x 10 ⁶There is the DMEM culture medium that does not contain FCS in the PR/8/34 virus of EID (50 μ l), or single culture base (being used for without infecting matched group) is cultivated.

After cultivating 60 minutes under 37 ℃, 200 μ l complete mediums are added to each hole, and plate cultivation under 37 ℃ is spent the night.Clean plate next day with PBS, and fix 10 minutes with 10% formalin under room temperature.Each hole is cleaned three times with PBS/0.1% BSA, and (BD Fa Minggen (BDPharmingen), Cat#555213), famous scenic spot tooth brother CA) in room temperature next hour, or spends the night under 4 ℃ and blockades with 200 μ l/ hole ADB.The serial dilution of test sera is added to each hole, and under room temperature, cultivated one to two hour.Resist-mouse IgG antibody (Jackson's immunochemistry with each hole cleaning and with the HRP-labelled goat, Cat115-035-146 (Xi Geluofu, PA)) cultivated 30 minutes under room temperature, subsequently with TMB Ultra substrate (Pierre Si (Cat#34028), Luo Kefu IL) cultivated two minutes under room temperature.With the 1N H of reaction with interpolation 25uL ₂SO ₄Stop, and (Dole breathes out, and NC) reads OD for FARCyte, Ai Moxun in using the micro plate spectrophotometer ₄₅₀

Result and discussion

In luring with STF2.HA1-2 (PR8) (SEQ ID NO:90) immunity back HA-specific IgG reaction Lead: STF2.HA1-2 exempt from different originality be by, in the 0th and 14 day the STF2.HA1-2 with a dosage range (30,3.0,0.3 or 0.03 μ g) test through subcutaneous immune BALB/c mouse (10/ group).Control group mice is to excite with 8x10 with the STF2.4xM2e of PBS (negative control group), 3 μ g (SEQ ID NO:94) (for the negative matched group of immunogenicity, exciting the positive matched group of efficacy study for causing death) or inferior causing death ¹The influenza separated strain PR/8/34 of EID causes and exempts to produce immune restoration phase animal.Rose (the 21st day) back 7 days in short, by the reaction of ELISA check HA-specific IgG.The result proves, causes with the STF2.HA1-2 (PR8) of 30,3 or 0.3 μ g and exempts from, and can induce unanimity and significant HA0sHis-specificity (SEQ ID NO:176) IgG to react (Fig. 6) by dose-dependent manner.

The STF2.HA1-2 (SEQ ID NO:90) that must hang oneself causes the serum of exempting from mice and is subjected to influenza infection Mdck cell reaction: aforesaid direct ELISA result proves, the serum of the STF2.HA1-2-immune animal of must hanging oneself can be discerned the HA0sHis (SEQ ID NO:176) of the expressed PR/8/34 of the being equivalent to HA of recombinant type fruit bat sequence.-HA antibody anti-in order to prove can be discerned natural viral HA, check then identical serum its with the ability of the mdck cell reaction that infected by PR/8/34.The result who is shown in Fig. 7 proves, the STF2.HA1-2 of 30,3 or 0.3 μ g of must hanging oneself causes the mice of exempting from, but specificity combines with the mdck cell that infected by PR/8/34, and expression exempts to act on the anti--HA antibody that is produced via causing with STF2.HA1-2, can discern the HA that exists with its native configurations.

Comprehensive these results prove, STF2.HA1-2 (PR8) through there being PBS (not having known adjuvant or supporting agent) (SEQ ID NO:90) causes the mice of exempting from, represent its can in vitro discern in fruit bat, express recombinant type HA, and be expressed in strong anti--HA reaction of the natural viral HA on the mdck cell surface that PR/8/34 infects.

Embodiment 6: the effect of representing the recombinant type dynein-hemagglutinin fused protein of Strain A/ Puerto Rico/8/34

Materials and methods

Influenza virus excites miceFor analyzing effect, then will be described through immune mouse as embodiment 5, gave LD in the 28th day by the intranasal throwing ₉₀(making the lethal dosage of 90% mice) (8x10 ³EID) influenza A separated strain PR/8/34 and exciting.Every day is investigated the survival of animal in the back, body weight runs off and clinical sign reaches 21 days in exciting.It is that meansigma methods with every group of animal out of the ordinary ((body weight every day (g)/28th day original body weight (g)) x100) is that basic calculation gets that the % body weight runs off.The appointment of clinical scores is as follows: 4 points=health, and 3 points=minimizing combing, 2 points=health is active to be lowered, and 1 point=dying.(experimental result that clinical scores and body weight run off reflects based on the result of surviving animals in assessed natural law).

Titration of virus and mensuration

Cell preparation: with mdck cell (ATCC (Cat# CCL-34), the Manassas, VA) be incubated at 100 x 20mm culture plates (BD (Cat# 353003) is healthy and free from worry, reach 90-95% in NY) to be paved with, and with monolayer by with trypsin-EDTA in 37 ℃/5% CO ₂Cultivated 20 minutes and break away from.By adding the DMEM cell culture medium that replenishes with 10%FBS trypsin is deactivated, and cell monolayer scraped with sterile spatula make the complete desorption of cell.Collect cell suspending liquid, and with twice of DMEM+10%FBS rinsing.The cell resuspending is also counted in DMEM+10%FBS.Cell concentration is adjusted to 4 x 10 ⁵Cell/ml, and 100 μ l are added to 96-hole tissue culturing plate, and (BD (Cat# 353075) is healthy and free from worry, in each hole NY).With plate in 37 ℃/5% CO ₂Cultivation is paved with up to reaching 90-95%.

Titration of virus: ((Luo Kelan (Cat#BSA-50) Ji Baisi paddy PA) is diluted to 1x10 to a stream part V in middle no phenol red DMEM+0.1%BSA with influenza virus (strain A/ Puerto Rico/8/34[PR/8]) ⁸EID, and use same medium in the 96-orifice plate, to be prepared into serial 5-times diluent.With the mdck cell monolayer that is prepared in the 96-orifice plate as described herein, by draining culture medium, displacement is drained PBS again with the 1x PBS in 200 μ l/ holes and is cleaned.With 5-times of diluent of influenza virus series of preparation as described above, add to this monolayer through cleaning with the volume in 100 μ l/ holes.Group is in contrast only handled with culture medium in the string hole.With cell in 37 ℃/5% CO ₂Cultivated 2 hours, and adhered to and enter to make virus.With each hole by drain, with 200 μ l/ hole PBS rinsings and drain PBS and clean.Make institute porose acceptance 100 μ l/ holes not have phenol red DMEM+0.1%BSA, and with plate in 37 ℃/5%CO ₂Cultivated 48 hours.

The mensuration of cell survival degree: after cultivating with virus as described above, culture medium is drained from each hole, and displacement is to contain 40 μ g/ml dimethyl diaminophenazine chlorides (inferior capable strange (Cat# N2889) the Saint Louis city of sigma, fresh culture MO).For measuring maximum dissolving, then 2 μ l solvent solns (9% Triton X-100 is water-soluble, weight/volume) are added to, only with three repeating holes of culture medium cultivation.After cultivating in one hour, by adding 100 μ l/ holes, 1% formaldehyde/1% CaCl ₂In following 5 minutes fixed cells of room temperature; This fixing step repeats twice and success.Drain through fixed solution, and 100 μ l/ holes extraction culture medium (50% ethanol/1% acetic acid) are added so that dimethyl diaminophenazine chloride is disengaged.

Plate was cultivated under room temperature 20 minutes, followed stirring in wherein last 2 minutes.By using the light absorption value of micro plate spectrophotometer measurement under 540mm, and measure the amount of dye of being disengaged.Measurement is compared to medium controls, and the dyestuff emission that is reduced is cell death (reaching therefore viral infection).When cultivate finishing, as described hereinly carry out the dimethyl diaminophenazine chloride analysis.The dissolving percentage ratio of each serum dilution is as follows as calculated:

% lowers=100 x ((sample-virus)/(med-virus))

Wherein sample, max and med mean and represent experiment sample respectively, only contain virus and only contain light absorption value in the hole of culture medium.It is to be defined as that the neutralization of each sample is tired, the serum dilution that causes 50% viral infection to lower.

Influenza A/PR/8/34 neutralization analysis: this analysis is to cooperate to be derived from the WHO handbook about the diagnosis of animal influenza and supervision, the p.86-88 modification of (WHO/CDS/CSR/NCS2002.5).The dimethyl diaminophenazine chloride analysis is to adapt to the open motion cover Wuerzburg institute Cell Lab (http://www3.gettysburg.edu/～sorense/Celllab04/neutralred.htm).

Especially, as previously mentioned with MDCK (ATCC (Cat#CCL-34), the Manassas, VA) the cell plain cloth in 96-hole tissue culturing plate (BD (Cat# 353075) is healthy and free from worry, NY) in.Test agent (experimental and control group serum) is passed through, in being heated to 56 ℃ water-bath, cultivated 30 minutes and through heat-deactivate.Serum in the 96-orifice plate, doubly is diluted among the no phenol red DMEM+0.1% BSA with 3-and carries out serial titration.Equal-volume is diluted in same medium to 5 x 10 ⁶The PR/8/34 virus of/ml EID, adding to each serum dilution is 2.5 x 10 to reach final virus concentration ⁶EID/ml (is the predetermined TCID of the present viral reserve of we ₅₀).Comprise the hole of only containing culture medium and only containing virus, respectively as feminine gender and positive controls.With plate in 37 ℃/5% CO ₂Cultivated 30 minutes.

Will be as described above ( Cell preparation) cell monolayer that is prepared into, clean once with 200 μ l/ hole 1x PBS, then with the prepared as described above serum in 100 μ l/ holes: virus mixture and matched group reagent add, and in 37 ℃/5% CO ₂Cultivated 2 hours.After cultivating, cell monolayer is cleaned once with PBS, and 100 μ l/ holes are not had phenol red DMEM+0.1% BSA add to each hole, and in 37 ℃/5% CO ₂Cultivated 48 hours.When cultivating end, (mensuration of cell survival degree) carries out the dimethyl diaminophenazine chloride analysis as mentioned before.Calculate the dissolving percentage ratio of each serum dilution as previously mentioned.It is to be defined as that the neutralization of each sample is tired, the serum dilution that causes 50% viral infection to lower.

Result and discussion

Provide with STF2.HA1-2 (PR8) (SEO ID NO:90) immunity and to avoid exciting with the deadly of influenza A Protective effect: the result by embodiment 5 proves that mice causes and exempts from STF2.HA1-2 (PR8) (SEQ ID NO:90), can produce the antibody response of the natural HA of identification.Be the assessment effect, satisfied in the 28th day and give LD through the intranasal throwing ₉₀(8 x 10 ³EID) PR/8/34 virus and exciting.Every day is investigated the survival of mice in the back, body weight runs off and clinical sign reaches 19 days in exciting.Shown in Fig. 8 A, 8B and 8C, cause the mice of exempting from through PBS-at early as after exciting three days, promptly present and infect symptom (body weight runs off and lower clinical scores), and all mices are in exciting back the 21st day all dead.Otherwise, cause the mice of exempting from through the STF2.HA1-2 of 30,3.0 or 0.3 μ g (PR8) (SEQ ID NO:90), prove remarkable enhanced protective effect.These animal proofs have less or do not have the body weight loss, and clinical scores is obviously higher, and is similar to immune restoration phase control animals, go through and still have 100% survival after 21 days.These results prove that through the STF2.HA1-2 of escherichia coli expression (PR8) (SEQ ID NO:90), inducing can be in vivo successfully protecting BALB/c mouse to avoid the deadly HA-specific immune response that excites of toxicity A type influenza virus.

Be the effectiveness in vitro of assessment antibody response, satisfy the viral neutralization analysis of development based on cell, with the test immune serum in order in and the ability of viral infection.In this research, be to check to such an extent that the STF2.HA1-2 that hangs oneself (PR8) (SEQ ID NO:90) and STF2.HA1-1 (PR8) (SEQ ID NO:151) cause the inhibition activity of the serum of exempting from animal.With the serial dilution of non-immunity and immune serum, cultivate in advance with PR/8, cultivate with the DUCK cell again.Each hole is cleaned removing free state virus, and dyeed with dimethyl diaminophenazine chloride after plate cultivated and detect living cells.The result points out that the STF2.HA1-2 (PR8) that must hang oneself (SEQ ID NO:90) causes the immune serum of exempting from animal, confirm to have tissue culture suppress 50% dosage (TCID) for 1:40 (Fig. 9).

Embodiment 7: the immunogenicity of representing the recombinant type dynein-hemagglutinin fused protein of Strain A/ Vietnam/1203/2004

Materials and methods

Zooscopy: use the 6-8 female BALB/c mouse in age in week (Jackson's laboratory, Ba Ergang, ME).With 10 every group of mice group, and accepted in the 0th and 14 day groin s.c cause exempt from as follows:

1) PBS (phosphate-buffered saline solution)

2) STF2.HA1-2 of 3.0 μ g (PR8) (SEQ ID NO:90) is in PBS

3) STF2.HA1-2 of 3.0 μ g (VN) (SEQ ID NO:95) is in PBS

4) STF2.HA1-2 of 0.3 μ g (VN) (SEQ ID NO:95) is in PBS

With mice in blood sampling in the 21st day, and with serum by condensing and centrifugal clarificationization, and store under-20 ℃.

Serum antibody is measured: measure HA and STF2-specific IgG concentration by ELISA.(Costar (Cat#9018) is healthy and free from worry with 96-hole elisa plate, NY) under 4 ℃, (BEI resource (BEI Resources) (Cat#NR-660) with HA albumen that 100 μ l/ holes get from Vietnam/1203/2004 purification, Meng Sasi, VA)), or reorganization flagellin (STF2) (SEQ ID NO:96) be dissolved in PBS (5 μ g/ml) and apply and spend the night.With the analysis dilution buffer liquid (ADB of plate with 200 μ l/ holes; BD Fa Minggen, (Cat#:555213) famous scenic spot tooth brother CA) blockaded under room temperature one hour.Plate is cleaned three times in the PBS (PBS-T) that contains 0.05% (v/v) Tween 20.Serum is added (100 μ l/ hole) in the diluent of ADB, and plate cultivation under 4 ℃ is spent the night.Plate is cleaned three times with PBS-T.With the HRP-labelled goat that is diluted in ADB anti--(Jackson's immunochemistry (Cat#:115-035-146), Xi Geluofu PA) add (100 μ l/ hole) to mouse IgG antibody, and plate was cultivated under room temperature 1 hour.Plate is cleaned three times with PBS-T.(IL), and behind the monitoring color colour generation, (Dole breathes out TMB Ultra substrate to be added, NC) measures A on the micro plate spectrophotometer in Tecan Farcyte for Pierre Si (Cat#34028), Luo Kefu ₄₅₀

Result and discussion

Detect the immunogenicity of STF2.HA1-2 (VN) (SEQ ID NO:95) in BALB/c mouse.With animal in the 0th and 14 day with 3 μ g STF2.HA1-2 (PR8) (SEQ ID NO:90), or cause through s.c. with 3 or 0.3 μ gSTF2.HA1-2 (VN) (SEQ ID NO:95) and to exempt from.In the 21st day animal is taken a blood sample, and detect for from the HA (available from BEI resource Cat#NR-660) that A/ Vietnam/1203/2004 purification gets, reach reorganization flagellin (STF2 by ELISA; The serum IgG reaction (Figure 10 A and 10B) of (SEQ ID NO:96).The result proves that must hang oneself causes the serum of the mice of exempting from, present with from the proteic antigen-specific reaction of H5 that A/ Vietnam/1203/2004 purification gets.

Embodiment 8: clone, performance and the biochemical characteristicsization of obtained recombinant type dynein-hemagglutinin fused protein in baculovirus

The clone: HA and STF2.HA Expression of Fusion Protein, be to use Bac-to-(because of the Vito gold, Ka Sibeide CA), finishes (Figure 11) according to the indication of manufacturer to Bac baculovirus (Baculovirus) expression system.The cDNA of coding HA and STF2.HA fusion rotein is cloned in the pFastBac donor plasmid, is used for transforming the DH10Bac cell that contains rod granule (bacmid) and auxiliary DNA then.Then will be identified by indigo plant-Bai screening of on the X-gal flat board, carrying out by the reorganization rod granule clone strain that homologous recombination produced that in DH10Bac, carries out.The details of clone's program are referring to following paragraph.

The generation of support card casket: pFastBac ^TM1 carrier can with the Bac-to-Bac baculovirus expression system (because of the Vito gold, Ka Sibeide, CA) compatible.This carrier has strong AcMNPV polyhedral body (PH) promoter for the high level protein expression, and for the big multiple cloning site of simplifying the clone.PFastBac ^TMThe 1st, a kind of non-fusion vector (that is, amixis label in this carrier).

For determining suitable express recombinant protein, insert must contain for the ATG start codon of translating initiation, and for the termination codon of translating termination.For helping to clone the flagellin that merges with several HA gene truncates, satisfy two kinds of plasmid cards of processing and fabricating casket: pFB-STF2Blp.wt (SEQ ID NO:97) and pFB-STF2Blp.ng (SEQ ID NO:98).In the latter, the supposition saccharifying site of flagellin (STF2) gene is lacked with Asn (N) by Gln (Q) is replaced.The mensuration in possible saccharifying site is to use the residue for N-glycosyl sequence N-X-S/T:N, and taking over what residue subsequently is S or T in the 3rd position afterwards.

In some specific site, the residue 40,122,215,237,414,478 and 497 of STF2 gene (SEQ ID NO:212) is to incorporate the N residue into to replace as Q.Hide these sudden changes, and the construct as if do not modified of protein wherein by saccharide, the non-saccharifying of called after (ng) saltant.Equally, the saccharifying sudden change is imported the position 11,23,268,286 and 480 (SEQ ID NO:23) of HA0s gene.This two vector constructions body (pFB-STF2Blp.wt and pFB-STF2Blp.ng) is all hidden silent mutation in the nucleotide GTGCTGAGCCTGTTACGT-3 ' of STF2 (SEQ ID NO:212) (SEQ ID NO:310) (nt 1501 to 1518), produce unique BlpI in plasmid card casket.Each blocks casket and contains the amino terminal that melittin (HBM) sequence (SEQ ID NO:99) is blended in STF2, so that secretion signal to be provided.Two plasmids all (authenticate reagent company, interior ground, TX through the codon optimization for being expressed in the B baculovirus interiorly; DNA2.0Inc covers Luo Gongyuan, CA).Synthetic gene is cut out with BamHI or BglII and SphI, and be cloned into pFastBac by compatible terminal the joint ^TMIn 1 carrier, produce pFB-STF2Blp.wt and pFB-STF2Blp.ng card casket.

Flagellin-HA fusion card casket: with the HA ball head subunit single expression of influenza virus strain A/ Puerto Rico/8/34, A/ Vietnam/1203/2004, A/ Indonesia/2005 and A/ New Caledonia/12/99, or on the gene level, merge to flagellin (STF2) (SEQ ID NO:178) and in baculovirus, express.This is with wherein a kind of finishing of following three kinds of methods:

Method #1: the position produces the reagent that is used for ELISA, and the HA1-1 subunit of satisfying PR8, VN, IND and NC strain (table 7 and table 8) is cloned into pFASTBac ^TMIn 1, produce the pFB.HA1-1 that it comprises six-His label.This is by using as the listed primer that shows of following table, with the synthesis type password-(DNA2.0 Inc., illiteracy Luo Gongyuan CA) carry out PCR and finish as dna profiling through optimization HA0s gene (HA gets rid of functional domain outside signal sequence and the born of the same parents).The PCR fragment is digested with BglII and SphI, and insert, subsequently the pFastBac that handles with BAP more in advance through BglII and SphI enzyme ^TMIn 1.For producing flagellin-HA subunit fusant, then the PCR product is digested with BlpI, and be engaged to pFB-STF2Blp.wt by compatible end.

Method #2: in this motion, be with chemosynthesis must represent wt and non-glycosylated form (DNA2.0Inc. covers Luo Gongyuan, CA) through codon-optimization HA subunit gene.Gene is gone out with BglII and SphI enzyme action, and will be engaged to, the pFB-STF2Blp.wt that handles through BglII and SphI and BAP, or non-saccharifying version (pFB-STF2Blp.ng) in advance through the fragment of gel-purified.In case out of the ordinary, be to use to engage mixture and transform the TOP10 cell, and transformant carried out screening with PCR and DNA sequencing, with determine insert exist with correct position to.Construct is used to transform MAX

DH10Bac ^TM(because of the Vito gold, Ka Sibeide CA), and produces the reorganization rod granule to competent escherichia coli.By on the flat board that contains 50 μ g/mL kanamycin, 7 μ g/mL gentamycins, 10 μ g/mL tetracyclines, 40 μ g/mLIPTG and Bluo-Gal (40. μ g/mL), carrying out indigo plant/white screening, go out positive rod granule from the pure strain screening of antibacterial.Preparation reorganization bacmid dna, and be used for changeing infection selected insect cell line (Sf9 or Sf21 cell), and produce the recombinant type baculovirus.Then the amplification of baculovirus reserve is reached and tire surely, and be used to infect High Five insect cell with express recombinant protein matter.

Table 7: be used for the PR8HA construct that baculovirus is expressed

SEQID NO：	Construct (His label)	Method	FOR primer SEQ ID NO:	REV primer SEQ ID NO:	Dna profiling SEQ ID NO:
						100	STF2.HA0s	#	1	101	102	103
104	STF2.HA1-1	#1	105	106	103
						107	STF2.HA1-2	#1	108	109	103
110	STF2.HA1-2mut	#2	N/A	N/A	111
						112	STF2.HA1-3	#1	113	114	103
115	STF2.HA1-3mut	#2	N/A	N/A	116
						117	ngSTF2.HA0s	#2	N/A	N/A	118
119	ngSTF2.HA1-1	#2	N/A	N/A	120
						121	ngSTF2.HA1-2	#2	N/A	N/A	122
123	ngSTF2.HA1-2mut	#2	N/A	N/A	124
						125	ngSTF2.HA1-3	#2	N/A	N/A	126
127	ngSTF2.HA1-3mut	#2	N/A	N/A	128
						129	wtSTF2.HA1-1ng	#2	N/A	N/A	130
131	ngSTF2.HA1-1wt	#2	N/A	N/A	132
						133	HA1-1	#1	134	135	136
137	HA1-1(no tag)	#1	134	138	136

Table 8: be used for the NC that baculovirus is expressed, VN and NC HA construct

SEQ ID NO：	Construct (His label)	Method	FOR primer SEQ ID NO:	REV primer SEQ ID NO:	Dna profiling SEQ ID NO:
						139	HA1-1(NC)	#1	140	141	142
143	HA1-1(VN)	#1	144	145	146
						147	HA1-1(IND)	#1	148	149	150

Protein expression: with meadow armyworm (Sf9) insect cell be incubated at Ge Shi insect cell culture medium (because of the Vito gold, Ka Sibeide, CA) in.The rod granule of will recombinating is purified into from the pure strain of DH10Bac, and use cell infection element (Cellfectin) (because of the Vito gold, Ka Sibeide, CA) commentaries on classics is infected to the Sf9 cell, produce the first generation (P1) baculovirus reserve, then it is tired according to fixed its of indication of manufacturer with traditional plaque analysis.Then using P1 virus reserve is 0.1 infection Sf9 cell with infection multiplicity (MOI), produces to have the P2 virus reserve that increases virus titer.To express following proteinic culture amplification culture, for protein purification: ngSTF2.HA (PR8) 1-2mutHis (SEQ ID NO:156); NgSTF2.HA (PR8) 1-1His (SEQ ID NO:152; STF2.HA (PR8) 1-1His (SEQ ID NO:151); STF2.HA (PR8) 1-2His (SEQ ID NO:153); STF2.HA1-2mutHis (SEQ IDNO:155); HA (PR8) 1-1His (SEQ ID NO:179) HA (VN) 1-1His (SEQ ID NO:180); HA (IND) 1-1His (SEQ ID NO:181); HA (NC) 1-1His (SEQ ID NO:182).By being 2 P2 viral infection with MOI, and under 28 ℃, follow slow shaken cultivation to carry out protein expression in 24 hours with the High-5 insect cell of 2x1L flask.By the centrifugal conditioned medium of collecting, and by by 0.22 μ m filter clarificationization and store in 4 ℃.

Protein purification: with NiSO ₄Adding to each supernatant to ultimate density through clarificationization is 0.5mM, and pH is adjusted to 8.0.To add His then ₆The protein capture of-label is in filling with NiSO ₄, and in equilibrated chelating agarose tubing string (the GE/ Ai Moxun bioscience of buffer A (20mM Tris, pH 8.0,0.5M NaCl); Skin SIKA tower prestige, NJ) on.Treat bonded protein to be carried out eluting with buffer B (buffer A+0.5M imidazoles) with after the buffer A flushing.Compile peak stream part, dialyse and to buffer, spend the night, and application of sample (sigma-Ya is capable strange to nickel NTA; Saint Louis city, MO) tubing string.Tubing string is carried out eluting with 5-tubing string volume from the linear gradient of 100% buffer A to 100% buffer B.Compile peak stream part, dialyse to 1X TBS, among the pH 8.0.

SDS-PAGE and western blotting: measure protein homogeneity and assessment purity by SDS-PAGE.The sample out of the ordinary of 5 μ g five equilibriums is diluted in, contains or do not contain in the SDS-PAGE sample buffer of 100mM DTT as Reducing agent.Sample is boiled 5 minutes, and application of sample to 10% sds page (LifeGels; The blue Si Senlin of method, Xin Nanweiersi, AUS) on, and carry out the electrophoresis art.With gel with the blue R-250 (Bio-Rad of Kao Maxi; Hull Ke Lishi, CA) dyeing is to present protein band.For Western blotting, be that the capable overall protein matter of 0.5 μ l/ is carried out electrophoresis as previously mentioned, through electricity-be transferred on the pvdf membrane, and blockade, then afterwards with anti--flagellin antibody (Inotek with 5% (w/v) milk powder; Bei Fuli, MA), anti--His ₆Antibody (because of the Vito gold, Ka Sibeide, CA), or influenza A PR/8/38 convalescent period immune serum (following described in proteantigen ELISA) is surveyed.Treat with alkali phosphatase-conjugated secondary antibodies (Pierre Si, Luo Kefu, IL) survey after, with alkali phosphatase colour generation substrate (Pu Meijia; Horse ground is gloomy, WI) presents protein band.

Protein analysis: (Pierre's Scirocco not IL) with the micro plate form, uses bovine serum albumin as reference material, according to the explanation of manufacturer, measures overall protein matter concentration to use Micro BCA (two cinchoninic acids) analysis.

Endotoxin analysis: use the QCL-1000 LAL test kit (Cambrex that quantitatively develops the color; E. the endotoxin degree not NJ), according to the explanation that manufacturer is used for the micro plate method, is measured in the Luther.

The TLR5 biological activity is analyzed: the HEK293 cell is expressed TLR5 inherently, and in the reaction to the TLR5 citation, secretes several soluble factors (comprising IL-8).With cell inoculation (50,000 cells/well) in the micro plate of 96-hole, and following test protein added and cultivate and spend the night: ngSTF2.HA (PR8) 1-2mutHis (SEQ ID NO:156); NgSTF2.HA (PR8) 1-1His (SEQID NO:152); STF2.HA (PR8) 1-1His (SEQ ID NO:151); STF2.HA (PR8) 1-2His (SEQ ID NO:153); STF2.HA1-2 (PR8) mutHis (SEQ ID NO:155).Next day, collect conditioned medium, be transferred to clean 96-hole micro plate, and freezing down in-20 ℃.After waiting to thaw, in the antibody that uses Anti-Human's class-IL-8 to cooperate to (Pierre Si, Luo Kefu, IL; #M801E and #M802B) sandwich ELISA, according to the explanation of manufacturer, the existence of IL-8 in the analysis condition culture medium.

Proteantigen ELISA: whether correctly show for measuring recombination fusion protein, then assess the construct of following purification from the baculovirus culture: ngSTF2.HA (PR8) 1-2mutHis (SEQ ID NO:156) by ELISA through folding HA epitope; NgSTF2.HA (PR8) 1-1His (SEQID NO:152); STF2.HA (PR8) 1-1His (SEQ ID NO:151); STF2.HA (PR8) 1-2His (SEQ ID NO:153); STF2.HA1-2mutHis (SEQ ID NO:155) and HA (PR8) 1-1His (SEQ ID NO:179).96-hole elisa plate with serial dilution each target protein (starting from 5 μ g/ml) in PBS (100 μ l/ hole), is applied under 4 ℃ and spends the night.With the analysis dilution buffer liquid (ADB of plate with 200 μ l/ holes; BD Fa Minggen), under room temperature, blockaded one hour, in PBS-T, clean three times then.Then the one-level antibody with fixed dosage adds to each hole.For analyzing the HA reactivity, to satisfy 100 μ l/ hole 1:10,000 non-immunity or the PR/8/34 convalescent period immune serum that is diluted in ADB adds.The PR/8/34 immune serum be in, the inferior deadly booster dose of accepting to get through measuring is 8 x 10 ¹The BALB/c mouse of the PR/8/34 influenza virus of the infectious dosage of egg (Jackson's laboratory, Ba Ergang, ME) the middle generation.Make animal after infection, recover then〉21 days, separate immune serum and clarificationization this moment.

For flagellin or the histidine-tagged ELISA of 6x, be (because of the Vito gold with anti-6x His; Ka Sibeide, CA), or flagellin (Inotek; Bei Fuli, monoclonal antibody MA) is dissolved in ADB (100 μ l/ hole) with 1 μ g/ml and adds, and plate was cultivated under room temperature 1 hour, or spends the night under 4 ℃.Then plate is cleaned three times with PBS-T.Resisting-mouse IgG antibody (Jackson's immunochemistry through HRP-labelling goat of ADB will be diluted in; Xi Geluofu PA) adds (100 μ l/ hole), and plate was cultivated under room temperature 1 hour.Plate is cleaned three times with PBS-T.TMB Ultra substrate to be added (Pierre Si, Luo Kefu, IL), and behind the monitoring color colour generation, in micro plate spectrophotometer (FARCyte, GE/ Ai Moxun bioscience; Skin SIKA tower prestige NJ) goes up measurement A ₄₅₀

Result and discussion

Reorganization proteic expression of HA and purity: the baculovirus expression result of reorganization STF2.HA fusion rotein is that integration column is shown in Table 9.All proteins all with in paramount output express.Purity after the metallo-chelate tomography is good substantially, and the endotoxin degree is far below the acceptable limits of 0.1EU/ μ g.All STF2 fusion rotein all prove effectively TLR5 activity in vitro of tool.

Table 9

Protein	SEQID NO：	Output (mg)	Purity est. (%)	Endotoxin (EU/ μ g)	TLR activity (EC 50，ng/ml)
						STF2.HA1-1His(PR8)	151	12	>80	0.01	3
ngSTF2.HA1-1His(PR8)	152	24	>90	<0.01	30
						STF2.HA1-2His(PR8)	153	13.2	>90	<0.01	9
STF2.HA1-2mutHis(PR8)	155	25	>95	<0.01	900
						ngSTF2.HA1-2mutHis(PR8)	156	6	>90	0.01	9

The albumen that will be used for baculovirus expression, processing have it and do not have N-and links the saccharifying site, translate the back modification to protein expression, folding and bioactive influence to measure this.NgSTF2.HA1-2.mutHis (PR8) (SEQ ID NO:156) (being non-saccharifying), as if with more corresponding glycated proteins height, its degree expression is in the baculovirus supernatant, and the expression saccharifying may influence this Protein Folding or secretion.As if yet this proteinic saccharifying and non-saccharifying version when analyzing by Western blotting, react with the convalescent serum that derives from influenza PR8-infecting mouse comparably.And the expression degree of ngSTF2.HA1-1His (PR8) (SEQ ID NO:152) is high than its saccharifying copy, and the expression saccharifying may be not easy by vague generalization for these proteinic influences.

Except protein, also analyze the HA antibody response of the small-scale baculovirus supernatant of STF2.HA1-3His (PR8) (SEQ ID NO:157) and STF2.HA1-3mutHis (PR8) (SEQ ID NO:158) with fairly large expression and purification.As observed to person (referring to embodiment #4) to be expressed in colibacillary STF2.HA1-3 (PR8) protein (SEQ ID NO:92), these protein are shown on the Western blotting of conditioned medium, with the reactive non-constant of PR8 convalescent serum.This result proves that further STF2.HA1-3 protein (no matter being expressed in protokaryon or eucaryon host) can suitably fold, and represents the native antigen epi-position.

Antigenicity: several STF2.HA protein that in baculovirus and escherichia coli, make by elisa assay.The all same and anti--flagellin antibody (Inotek of all STF2 fusion rotein; Bei Fuli, MA) reaction is good, and expression equates that the protein application of sample is in this is analyzed.Then by measuring the reaction of each protein and the mice serum that has recovered from the PR/8/34 influenza infection, check the suitably folding of antibody antigen epi-position in the STF2.HA fusion rotein and show.STF2.HA1-1His (PR8) (SEQID NO:151), ngSTF2.HA1-1His (PR8) (SEQ ID NO:152) that baculovirus is expressed and ngSTF2HA1-2mutHis (PR8) (SEQ ID NO:156) and the expressed STF2.HA1-2 of escherichia coli (PR8) (SEQ ID NO:90) (referring to embodiment #4) are suitable with the reactivity of PR/8/34 convalescent serum.Therefore, serum antibody identification HA ball head may concentrate on the zone that is comprised by HA1-2.Moreover, in vitro folding in the obtained STF2.HA1-2 of escherichia coli (SEQ ID NO:90), produces a kind of its have with from the protein of eucaryon host (baculovirus) through processing and oozy proteinic immunoreactivity suitable (and so folding configuration similar).In addition, the identification of the serum antibody of HA ball head may seem and not be dependent on saccharifying, and (at least for PR/8/34HA) may not influenced by saccharifying negatively.The HA (hemagglutinin) that is derived from different strains of influenza viruses has various different N-binding glycosylated form.Saccharifying may negatively influence the immunity identification of other HAs.The serum antibody identification of HA is not influenced by the N-saccharifying can.May be that not so saccharifying can influence effectiveness, for example half-life of vaccine.

Embodiment 9: the immunogenicity of the recombinant type dynein-hemagglutinin fused protein that makes in baculovirus

Materials and methods

Zooscopy: use the 6-8 female BALB/c mouse in age in week (Jackson's laboratory, Ba Ergang, ME).With 10 every group of mice group, and near accepting groin in the 0th and 14 day subcutaneous (s.c) cause exempt from as follows:

1) PBS (phosphate-buffered saline solution)

2) STF2.HA1-2 of 3 μ g (PR8) escherichia coli (SEQ ID NO:90) are in PBS

3) STF2.HA1-1 of 3 μ g (PR8) baculovirus (SEQ ID NO:151) is in PBS

4) ngSTF2.HA1-1 of 3 μ g (PR8) baculovirus (SEQ ID NO:152) is in PBS

5) ngSTF2.HA1-1 of 0.3 μ g (PR8) baculovirus (SEQ ID NO:152) is in PBS

6) STF2.HA1-1 of 3 μ g (PR8) escherichia coli (SEQ ID NO:89) are in PBS

7) STF2.HA1-1 of 0.3 μ g (PR8) escherichia coli (SEQ ID NO:89) are in PBS

With mice (the short liter) blood sampling in the 10th day (elementary) and 21 days, and with serum by condensing and centrifugal clarificationization, and store under-20 ℃.To give LD in the 28th day by the intranasal throwing through immune mouse ₉₀(8 x 10 ³EID) PR/8/34 virus and excite (referring to embodiment 6).Every day is investigated the survival of animal in the back, body weight runs off and clinical sign reaches 21 days in exciting.

Serum antibody is measured: measure HA-specific IgG concentration by ELISA.(Costar (Cat #9018), healthy and free from worry, NY) under 4 ℃, the HA0sHis protein of making in fruit bat with 100 μ l/ holes (SEQ ID NO:176) is dissolved in PBS (5 μ g/ml) and applies and spend the night with 96-hole elisa plate.With the analysis dilution buffer liquid (ADB of plate with 200 μ l/ holes; BD Fa Minggen, (Cat#:555213), famous scenic spot tooth brother CA) blockaded under room temperature one hour.Plate is cleaned three times in containing 0.05% (v/v) Tween, 20 (PBS-T).Serum is added (100 μ l/ hole) in the diluent of ADB, and plate cultivation under 4 ℃ is spent the night.Plate is cleaned three times with PBS-T.The HRP-labelled goat that is diluted in ADB is resisted-mouse IgG antibody (Jackson's immunochemistry (Cat#:115-035-146)) adding (100 μ l/ hole), and plate was cultivated under room temperature 1 hour.Plate is cleaned three times with PBS-T.(PA), and behind the monitoring color colour generation, (Dole breathes out TMB Ultra substrate to be added, NC) measures A on the micro plate spectrophotometer in Tecan Farcyte for Pierre Si (Cat# 34028), Luo Kefu ₄₅₀

Result and discussion

Be expressed in immunogenicity and the effect of the STF2.HA1-1 (PR8) of escherichia coli and baculovirus: BALB/c mouse caused through s.c. with the specified recombinant protein of 3.0 or 0.3 μ g in the 0th and 14 day exempt from.In the 21st day with animal blood sampling, and the ELISA detection HA-specific IgG of the HA0sHis protein (SEQ ID NO:176) of expressing by the antagonism fruit bat is tired.Figure 12 A, 12B shows described protein induce HA-specific IgG in various degree, and wherein ngSTF2.HA1-1 (PR8) (SEQ ID NO:152) induces and causes and observe the strongest similar reaction in the animal of exempting to be expressed in colibacillary STF2.HA1-2 (PR8) (SEQ ID NO:90).Interesting ground, STF2.HA1-1 (PR8) (SEQ ID NO:151) through contain complete saccharifying sequence in baculovirus expression causes few to the antagonism HA that can't detect or the antibody of flagellin, obviously with wherein jointly the removed STF2.HA1-1 of saccharifying sequence (PR8) (SEQ IDNO:152) to cause in the animal of exempting from viewed result equally opposite.

To give LD in the 28th day by the intranasal throwing through immune mouse ₉₀(8 x 10 ³EID) PR/8/34 virus and exciting.Every day is investigated the survival of animal in the back, body weight runs off and clinical sign reaches 21 days in exciting.Shown in Figure 13 A, 13B, 13C; cause the mice of exempting from through PBS-at early as after exciting three days; promptly present and infect symptom (body weight runs off and lower clinical scores); and it is promptly dead before the 21st day; and cause the mice of exempting from through 3.0 μ g reorganization STF2.HA1-2 (PR8) (SEQ ID NO:90) fusion rotein; because of less or do not have clinical symptom or the body weight observe infection and run off, and proof has enhanced protective effect.Though causing the animal of exempting from through the STF2.HA1-1 fusion rotein proves, it causes the animal of exempting from through independent PBS and has significantly higher effect, but these animals are compared to the animal of accepting STF2.HA1-2 (PR8) (SEQ ID NO:90), aspect pure work, clinical scores and TBW loss, present lower effect.These results prove, HA subunit in the baculovirus binding to STF2 is an immunogenicity and effective, and hinting the STF2.HA1-1 (SEQ ID NO:89) that is expressed in escherichia coli or baculovirus, the STF2.HA1-2 (SEQ ID NO:89) that is expressed in escherichia coli or baculovirus has relatively poor immunogenicity and more invalid.

The purification of embodiment 10:STF2.HA1-2 (IND) (SEQ ID NO:159)

Materials and methods

The cell deposit: will be incubated in the exclusive MRSF culture medium in order to express the Bacillus coli cells of STF2.HA1-2 (IND) (SEQ ID NO:159), to adapt to through operation, the glycerol reserve saves as the 1mL equal portions, and stores in-70 ℃.

The MRSF culture medium, pH7.0

Form G/L

Glucose 10

KH ₂PO ₄ 7.8

(NH ₄) ₂SO4 2.33

Citric Acid 1.0

MgSO ₄(7H ₂0) 1.0

CaCl ₂ 0.04

Trace meter 1ml

Thiamine HCl 0.01

Kanamycin 0.0075

Trace meter solution 1000x

Form G/L

EDTA, disodium 5

FeSO ₄(7H ₂O) 10

ZnSO ₄(7H ₂O) 2

MnSO ₄(H ₂O)2

CoCl ₂(6H ₂O)0.2

CuSO ₄(5H ₂O) 0.1

Na ₂MoO ₄(2H ₂O)0.2

H ₃BO ₃ 0.1

H ₂O 1000ml

The cell scale enlarges: with two bottles (2ml lays in the MRSF culture medium) through operation in order to the Bacillus coli cells of expressing STF2.HA1-2 (IND) (SEQ ID NO:159) from-70 ℃ of recoveries, and be seeded in the MRSF culture medium of 500ml.Cell was increased OD at that time in 11.5 hours by cultivating in-37 ℃ ₆₀₀Be 3.2.The cell scale enlarges can be provided for inoculation, uses the biological quality at the production bioreactor of fermentation.

Fermentation: 250mL through the cell that scale enlarges, is used to inoculate the bioreactor that 12L effect volume contains the special-purpose MRBR culture medium of 10L.Culture is followed constant agitation, keep pH value simultaneously in 7.0 ± 0.1, temperature is in 30 ± 0.1 ℃, and dissolved oxygen amount (DO) cultivates in being not more than 30%, and carries out till glucose exhausts with a batch part pattern.It is with desired O that glucose exhausts ₂Be reduced to index suddenly, and by selecting the glucose meter measure glucose concentration to confirm in YSI 2700.This moment culture OD ₆₀₀Be 20.1AU.

Exhaust back 30 minutes in glucose, feeding is cultivated based on squeezing in the bioreactor till adding the 2L feeding with the group Pu under the controlled velocity.Continue down to cultivate 5.5 hours in glucose-restrictive condition.In OD ₆₀₀During for 34AU, be 2mM, and follow constant agitation to cultivate 2 hours by IPTG being added to final concentration, and the protein expression in the inducing culture thing.When induction period finishes,,, collected cell under the 000g in centrifugal 20 minutes in 10 by in Avanti JP-20XP centrifuge.The final OD of culture ₆₀₀Be 58AU, and by BCA measure overall protein matter concentration be 6.73mg/mL.From 10.8L collect thing reclaim the 1.16kg cell precipitation, and down freezing in-20 ℃.Total bioreactor operation time is 17 hours.Determine that by SDS PAGE STF2.HA1-2 (IND) (SEQ ID NO:159) makes.

The MRBR culture medium, pH 7.0

Form G/L

Glucose 20

KH ₂PO ₄ 2.2

(NH ₄) ₂SO ₄ 4.5

Citric acid 1.0

MgSO ₄(7H ₂0) 1.0

CaCl ₂ 0.04

Trace meter 1ml

Thiamine HCl 0.01

Defoamer 0.05

Kanamycin 0.0075

The feeding culture medium, pH6.0-be added into MRBR culture medium (2L final volume)

Form G/L

Glucose 160

KH ₂PO ₄ 5.6

DL-alanine 50.0

Citric acid 3.0

MgSO ₄(7H ₂0) 1.0 (adding) with 78g/L solution

CaCl ₂(2.5 adding) with 80g/L solution

Trace meter 24ml

FeSO ₄7H ₂O 0.75

Cytoclasis and clarificationization: cell precipitation is thawed, and resuspending becomes 50mM Tris, 25mMNaCl, 15% suspension of pH8.In the APV1000 homogenizer, in 12,000psi homogenizes for following three times with serosity.Lysate is adjusted to pH4.0 by adding acetic acid, and centrifugal to separate solubility and insolubility material.STF2.HA1-2 (IND) (SEQ ID NO:159) is dispensed to the insolubility part with other protein.Solable matter is abandoned, and insolubility material (precipitation) is dissolved in the 50mM acetic acid of the original lysate volume of twice, 1% TritonX-100,8M carbamide is among the pH 4.To be mixed in the homogenizer of 0psi, sample is centrifugal and collect supernatant, filter and store under 4 ℃.Determine the existence of STF2.HA1-2 (IND) (SEQ ID NO:159) in the suitable phase (precipitation or supernatant) by SDSPAGE.

Cation-exchange chromatography art (CEX): will be through the material application of sample of clarificationization to 50ml Poros 50HS tubing string.This step is that design is used so that eluate compared to the application of sample thing, can reduce endotoxin concns, reduces nucleic acid concentration and increases STF2.HA1-2 (IND) (SEQ ID NO:159) purity.After treating application of sample, with the 50mM acetic acid of tubing string with five tubing string volumes, 1% Triton X-100,8M carbamide, pH 4, and subsequently with the 50mM acetic acid of 10 tubing string volumes, 8M carbamide, pH 4 flushings.With protein in to the 0-100% gradient of 1M NaCl, carrying out eluting.Target protein is that eluting goes out under about 300mM NaCl.Compile main peak (eluate) and store under 4 ℃.Determine the existence of STF2.HA1-2 in the eluent (IND) (SEQ ID NO:159) by SDS PAGE.In comparison application of sample material and this eluent, the number of protein band and intensity and measure the purification of STF2.HA1-2 (IND) (SEQ ID NO:159) through this step.

In cation-exchange chromatography art (CEX), remove endotoxin: endotoxin is with 50mM acetic acid, 1%Triton X-100, and 8M carbamide, pH 4 cleans and suitable elution requirement, removes from eluting STF2.HA1-2 (IND) (SEQ ID NO:159).This is to use STF2.HA1-2 (VN) (SEQ ID NO:95) test.Two bouts are comprised and are not contained 50mM acetic acid, 1% Triton X-100,8M carbamide, the cation exchange operation (Toso SP650M resin) of pH 4 flushings (the ER flushing is washed no ER) compares, and the endotoxin concns under 150mMNaCl in the ER flushing eluent shows, obviously than the 250mmNaCl eluent from identical bout, or in not containing 50mM acetic acid, 1% Triton X-100,8M carbamide, the endotoxin concns of the eluent of pH 4 flushings is few.

Eluent [endotoxin] [endotoxin] [protein] passes through UV

Bout

(mM aCl) (EU/mL) (EU/mg) BCA(g/L) 280/260

Load N/A 4.4 x 10 ⁶1.8 x 10 ⁶2.39-

100 7.9 x 10 ⁶ 4.2 x 10 ⁶ 1.44 1.05

No ER flushing

250 8.0 x 10 ⁶ 4.1 x 10 ⁶ 1.12 2.76

150 5，112 2，888 1.77 0.982

The ER flushing

250 24，440 N/A N/A 0.545

Folding again: with the CEX eluate to 50mM Tris, 8M carbamide, pH 8 buffer dialysis, and undertaken again folding by rapid dilution to 10 volume.

Anion-exchange chromatography art (AEX): will be through folding again protein application of sample to 25ml GE sucrose Q tubing string.This step is design in order to will separating with aggregation and host cell proteins matter through suitably folding STF2.HA1-2 (IND) (SEQ ID NO:159), and lowers the endotoxin concns of eluate compared to the application of sample thing.Behind application of sample, with the 50mM Tris of tubing string with five tubing string volumes, 25mM NaCl, pH8 flushing.With protein in to the 0-100% gradient of 1M NaCl, carrying out eluting.Eluting goes out to be rich in through suitably folding STF2.HA1-2 (IND) (SEQ ID NO:159) under about 150mM NaCl, and contains the peak of low endotoxin (1.7EU/mL).The protein that other comprises STF2.HA1-2 (IND) (SEQ ID NO:159) aggregation is contained on second peak that eluting goes out under about 170 to 270mM NaCl.Compile the stream part in first peak, and store under 4 ℃.Keeping the stream part that derives from first peak handles for further.Determine the existence of STF2.HA1-2 in first and second eluent (IND) (SEQ ID NO:159) by SDSPAGE.In comparison application of sample material and this eluent, the number of protein band and intensity and measure the purification of STF2.HA1-2 (IND) (SEQ ID NO:159) through this step.

The preparation molded dimension is got rid of tomography (SEC): the AEX eluate is flowed part to TBS with 2 x 5mL, on 320ml SEC tubing string, circulate.This step is design in order to will be through correct folding STF2.HA1-2 (IND) (SEQ ID NO:159) and other Separation of Proteins.Each result that circulates produces the eluate on single main peak, with its collection, compile and by 0.22 μ m filter.Product is stored under-70 ℃.

Determine the existence of STF2.HA1-2 in this eluent (IND) (SEQ ID NO:159) by SDS PAGE.In comparison application of sample material and this eluent, the number of protein band and intensity and measure the purification of STF2.HA1-2 (IND) (SEQ ID NO:159) through this step.Final species distribution on SDS PAGE is a strong single band.

Result and discussion

Proteinic final characterization: reorganization STF2.HA1-2 (IND) (SEQ ID NO:159) albumen is in expression in escherichia coli, and purified to homogeneity.With end product allocate in 1x TBS (27.7mM Tris, 2.7mM KCl, 137mM NaCl, pH 7.4; Make Teknova catalogue #T9530 from the 10x reserve), and store under-70 ℃ with the 1.0ml equal portions.Ultimate output is 5.75ml (45ml concentration is 0.35mg/ml), and wherein the endotoxin content that records with the LAL method is a 1.1EU/mg protein.When disengaging in cytokine when measuring in the analysis, still possess the TLR5 biological activity based on cell.

SDS-PAGE: measure protein homogeneity and assessment purity by SDS-PAGE.One five equilibrium sample is diluted in SDS-PAGE sample buffer and the diluent.Sample is boiled 5 minutes, and application of sample to 4 is to 12% sds page (because of Vito gold NuPage), and carries out the electrophoresis art.Gel is dyeed with the blue R-250 of Kao Maxi, to present protein band.By being positioned at the band that BioRad precision on the identical gel adds about 75kDa place of reference material, point out the positive findings of STF2.HA1-2 (IND) (SEQ ID NO:159).Compare STF2.HA1-2 (IND) (SEQ ID NO:159) its purity of strength assessment by range estimation with respect to all other bands.

The TLR5 biological activity is analyzed: the HEK293 cell is expressed TLR5 inherently, and in the reaction to the TLR5 citation, secretes several soluble factors (comprising IL-8).With cell inoculation (50,000 cells/well) in the micro plate of 96-hole, and test protein added and cultivate and spend the night.Next day, collect conditioned medium, be transferred to clean 96-hole micro plate, and freezing down in-20 ℃.After waiting to thaw, in the antibody that uses Anti-Human's class-IL-8 to cooperate to (Pierre Si, Luo Kefu, IL; #M801E and #M802B) sandwich ELISA, exist according to the IL-8 in the explanation analysis condition culture medium of manufacturer.(FARCyte Ai Moxun) measures optical density (OD) to use the micro plate spectrophotometer.

Endotoxin analysis: use the QCL-1000 LAL test kit (Cambrex) that quantitatively develops the color,, measure endotoxin concns according to the explanation that manufacturer is used for the micro plate method.

Protein analysis: use Micro BCA (two cinchoninic acids) to analyze (Pierre Si), use bovine serum albumin,, measure overall protein matter concentration according to the explanation of manufacturer as reference material with the micro plate form.

UV280/260 analyzes: this analyzes to measuring in the sample, compared to the nucleic acid concentration of protein concentration.The maximum light absorption value of nucleic acid betides about 260nm, and proteinic maximum light absorption value betides about 280nm.Light absorption value in 280nm is higher for the light absorption value ratio of 260nm, and expression sample rich in proteins is more than nucleic acid.

Embodiment 11: clone, performance and the biochemical characteristicsization of obtained recombinant type dynein-hemagglutinin fused protein in fruit bat

Materials and methods

HA is in clone and the expression of fruit bat (Drosophila): (Kui Erjin Cat#57704), according to the explanation of manufacturer, isolates RNA from influenza A strain A/ Puerto Rico/8/34 to use the centrifugal test kit of QIAamp MinElute virus.Use RT to use first burst of synthesis system of SuperScript III (because of the Vito gold; Cat#18080-051), or the SuperScript III one step RT-PCR system with the high correctness of platinum-Taq (because of the Vito gold; Cat# 12574-030), with HA RNA reverse transcription, and use TOPO cloning vehicle PCRII Zero Blunt (because of the Vito gold) to clone the cDNA according to the explanation of manufacturer.Then with HA0s fragment (not containing signal sequence and the HA gene of striding the film functional domain), the strategy that uses PCR-based is in the C-of card casket end and NH2 end, time cloning is gone in the pMT/STF2 Δ, and produces construct STF2 Δ .HA0s (PR8) (SEQ ID NO:160) and HA0s (PR8) .STF2 Δ (SEQ ID NO:167) respectively.Also the HA gene clone is gone into not contain in the pMT/Bip/V5-his carrier of STF2 Δ, and produce construct HA0s.his (SEQ ID NO:170).Table 10 lists described construct and is used to make up they's primer.

Table 10: be used for the PR8HA construct that fruit bat is expressed

SEQID NO：	Construct	FOR primer SEQ ID NO:	REV primer SEQ ID NO:	Dna profiling SEQ ID NO:
					160	STF2Δ.HA0sHis	161	162	163
164	STF2Δ.HA0s	165	166	163
					167	HA0s.STF2Δ	168	169	163
170	HA0sHis	171	172	163
					173	HA0s	174	175	163

Recombinant protein is from the expression of fruit bat Dmel-2 cell: with HA0s.His (SEQ ID NO:170) and STF2 Δ .HA0s (SEQ ID NO:164) plasmid, be total to the pCoBlast plasmid-change and infect to fruit bat Dmel-2 cell, and produce Dmel-2 HA0sHis (PR8) and Dmel-2 STF2 Δ .HA0s (PR8) stabilized cell.The Dmel-2 stabilized cell is expanded to from sticking together culture, and [fruit bat Sfm culture medium is (because of the Vito gold the selection culture medium; Ka Sibeide, CA)+25 μ g/ml blasticidin] shake culture, be extended to the 12L production scale then.

By adding CuSO ₄To ultimate density be 0.5mM, induce recombinant protein expression.After cultivating 72 hours, cell is removed from conditioned medium with centrifugal.Then conditioned medium is passed through 0.22 μ m filter clarificationization, and store under 4 ℃.

The purification of HA0sHis (PR8) (SEQ ID NO:176): with the conditioned medium application of sample to filling with NiSO ₄, and in equilibrated chelating agarose tubing string (the GE/ Ai Moxun bioscience of buffer A [20mM Tris, pH 8.0,0.5M NaCl]; Skin SIKA tower prestige NJ), and is carried out eluting in the linear gradient with buffer B [buffer A+0.5M imidazoles].Albumen is passed through in cushioning saline solution (TBS), pH 8.0 equilibrated Superdex 200 SEC tubing strings (GE/ Ai Moxun bioscience through 1X Tris-; Skin SIKA tower prestige is carried out fraction on NJ) and is further purified.Compile HA0s.His ₆(PR8) stream part, five equilibrium also stores in-80 ℃.

SDS-PAGE and western blotting: measure protein homogeneity by SDS-PAGE, and assessment purity.The sample out of the ordinary of 5 μ g five equilibriums is diluted in, contains or do not contain in the SDS-PAGE sample buffer of 100mM DTT as Reducing agent.Sample is boiled 5 minutes, and application of sample to 10% sds page (LifeGels; The blue Si Senlin of method, Xin Nanweiersi, AUS) on, and carry out the electrophoresis art.With gel with the blue R-250 (Bio-Rad of Kao Maxi; Hull Ke Lishi, CA) dyeing is to present protein band.For Western blotting, be that the capable overall protein matter of 0.5 μ l/ is carried out electrophoresis as previously mentioned, through electricity-be transferred on the pvdf membrane, and blockade, then afterwards with anti--flagellin antibody (Inotek with 5% (w/v) milk powder; Bei Fuli, MA), or influenza A PR/8/38 convalescent period immune serum (following described in proteantigen ELISA) is surveyed.Treat that (IL) after the detection, (Pu Meijia, horse ground is gloomy, WI) presents protein band with alkali phosphatase colour generation substrate for Pierre Si, Luo Kefu with alkali phosphatase-conjugated secondary antibodies.

Protein analysis: use Micro BCA (two cinchoninic acids) to analyze (Pierre Si, Luo Kefu IL), use bovine serum albumin,, measure overall protein matter concentration according to the explanation of manufacturer as reference material with the micro plate form.

Endotoxin analysis: use the QCL-1000 LAL test kit (Cambrex that quantitatively develops the color; E. the endotoxin degree not NJ), according to the explanation that manufacturer is used for the micro plate method, is measured in the Luther.

The TLR5 biological activity is analyzed: the HEK293 cell is expressed TLR5 inherently, and in the reaction to the TLR5 citation, secretes several soluble factors (comprising IL-8).With cell inoculation (50,000 cells/well) in the micro plate of 96-hole, and the reorganization fruit bat conditioned medium that will contain HA0sHis (PR8) (SEQ ID NO:176) or STF2 Δ .HA0s (PR8) (SEQ ID NO:177) adds.Next day, collect conditioned medium, be transferred to clean 96-hole micro plate, and freezing down in-20 ℃.After waiting to thaw, in the antibody that uses Anti-Human's class-IL-8 to cooperate to (Pierre Si, Luo Kefu, IL; #M801E and #M802B) sandwich ELISA exists according to the IL-8 in the explanation analysis condition culture medium of manufacturer.Use micro plate spectrophotometer (FARCyte, GE/ Ai Moxun bioscience; Skin SIKA tower prestige NJ) is measured optical density (OD).

Proteantigen ELISA: by the purified HA0s.His of ELISA test ₆Whether (PR8) (SEQ IDNO:176) shows through correct folding HA epitope to measure recombinant protein.With 96-hole elisa plate with the HA0s.His of serial dilution in PBS (100 μ l/ hole) ₆(PR8) (SEQ ID NO:176) protein (starting from 5 μ g/ml) applies under 4 ℃ and spends the night.With the analysis dilution buffer liquid (ADB of plate with 200 μ l/ holes; BD Fa Minggen), under room temperature, blockaded one hour, in PBS-T, clean three times then.Then the one-level antibody with fixed dosage adds to each hole.Be to analyze the HA reactivity, satisfy 100 μ l/ holes 1: 10,000 non-immunity or the PR/8/34 convalescent period immune serum that is diluted in ADB adds.The PR/8/34 immune serum be in, the inferior deadly booster dose of accepting to get through measuring is 8 x 10 ¹The BALB/c mouse of the PR/8/34 influenza virus of the infectious dosage of egg (EID) (Jackson's laboratory, Ba Ergang, ME) the middle generation.

Make animal after infection, recover then〉21 days, separate immune serum and clarificationization this moment.For the histidine-tagged ELISA of 6x, be (because of the Vito gold with anti-6x His; Ka Sibeide, CA), or flagellin (Inotek; Bei Fuli, monoclonal antibody MA) is dissolved in ADB (100 μ l/ hole) with 1 μ g/ml and adds, and plate was cultivated under room temperature 1 hour, or spends the night under 4 ℃.Then plate is cleaned three times with PBS-T.The HRP-labelling goat that is diluted in ADB is resisted-mouse IgG antibody (Jackson's immunochemistry; Xi Geluofu PA) adds (100 μ l/ hole), and plate was cultivated under room temperature 1 hour.Plate is cleaned three times with PBS-T.TMB Ultra substrate to be added (Pierre Si, Luo Kefu, IL), and behind the monitoring color colour generation, micro plate spectrophotometer (FARCyte, GE/ Ai Moxun bioscience; Skin SIKA tower prestige NJ) is measured A ₄₅₀

Result and discussion

The characterization of the HA0s of fruit bat-expressed: conditioned medium is with anti--His ₆Antibody is (because of the Vito gold; Ka Sibeide, western blot analysis CA) is determined the expression of HA0sHis (PR8) (SEQ ID NO:176), and with anti--flagellin antibody (Inotek; Bei Fuli, western blot analysis MA) determines that STF2 Δ .HA0s (PR8) (SEQ ID NO:177) is by the expression of changeing the fruit bat Dmel-2 cell that infects through corresponding expression vector.All by being non-reduced form, with the Western blotting identification of mice PR/8/34 convalescent period immune serum, and protein is promptly eliminated identification to this two protein after the DTT reduction.This result points out that this two secretary protein has correct disulfide bond.Conditioned medium represents significantly TLR5 activity in vitro to STF2 Δ .HA0s (PR8) (SEQ ID NO:177), and HA0sHis ₆(PR8) (SEQ ID NO:176) culture medium then denys (as expected).Pass through ELISA, purified HA0sHis at last ₆(PR8) (SEQ ID NO:176) albumen shows, itself and PR/8/34 convalescent period immune serum have remarkable reactivity.These results represent that HA0s protein is with through suitably folding form, goes out from fruit bat Dmel-2 emiocytosis.

Embodiment 12: immunogenicity and the effect of representing the recombinant type dynein-hemagglutinin fused protein of Strain A/ Vietnam/1203/04

Materials and methods

Zooscopy: use the 6-8 female BALB/c mouse in age in week (Jackson's laboratory, Ba Ergang, ME).With 15 every group of mice group, and accepted in the 0th and 14 day subcutaneous (s.c) cause exempt from as follows:

PBS (phosphate-buffered saline solution)

Do not have to cause and exempt from

The STF2.HA1-2 of 10 μ g (VN) (SEQ ID NO:95) is in PBS

3.0 the STF2.HA1-2 of μ g (VN) (SEQ ID NO:95) is in PBS

The STF2.HA1-2 of 1 μ g (VN) (SEQ ID NO:95) is in PBS

With mice in blood sampling in the 21st day, and with serum by condensing and centrifugal clarificationization, and store under-20 ℃.

Serum antibody is measured: measure HA-specific IgG concentration by ELISA.(Costar (Cat#9018) is healthy and free from worry, NY) under 4 ℃, derives from the HA protein of Vietnam/1203/04 with 100 μ l/ holes, makes in baculovirus, and (BEIR catalog number NR-660) is stored in PBS (1 μ g/ml) and applies and spend the night with 96-hole elisa plate.With the analysis dilution buffer liquid (ADB of plate with 300 μ l/ holes; BD Fa Maming, (Cat#:555213) (famous scenic spot tooth brother CA) blockaded under 25 ℃ two hours.Plate is cleaned three times in PBS+0.05% (v/v) Tween20 (PBS-T).Serum is added (100 μ l/ hole) in the diluent of ADB, and plate was cultivated 1.5 hours down in 25 ℃.Plate is cleaned three times with PBS-T.The HRP-labelled goat that is diluted in ADB is resisted-mouse IgG antibody (Jackson's immunochemistry, Xi Geluofu, PA (Cat#:115-035-146)) adding (100 μ l/ hole), and plate was cultivated 30 minutes down in 25 ℃.Plate is cleaned three times with PBS-T.(IL), and behind the monitoring color colour generation, (molecular device, the sun paddy CA) are measured A on the micro plate spectrophotometer to TMB Ultra substrate to be added in SpectraMax 190 for Pierre Si (Cat 34028), Luo Kefu ₄₅₀

Influenza virus excites mice: for analyzing effect, satisfy mice was given 10LD in the 28th day by the intranasal throwing ₉₀(10x makes the lethal dosage of 90% mice) (6 x 10 ³EID) influenza A/ Vietnam/1203/04 and exciting.Every day is investigated the survival of animal in the back, body weight runs off and clinical sign reaches 21 days in exciting.

The preparation of influenza H5N1 reserve: influenza A/ Vietnam/1203/04 (H5N1) be derive from disease control and prevention center (Atlanta, GA).Contained 10-days ages and to be prepared into viral reserve in embryo's egg, and be divided into the 0.5ml five equilibrium and store in-80 ℃ up to use.

The mensuration of virus titer: utilize to be inoculated in the 96-orifice plate, and grow to the mdck cell mensuration TCID that is paved with ₅₀(50% TCID).With a series of viral log ₁₀Diluent is inoculated on the quadruple compound plate.Plate is cultivated 48-72 hour then.By determining to make the dilution factor that 1/2 positive and 1/2 negative viral growth arranged in the multiple orifice plate of quadruple, and measure TCID ₅₀EID ₅₀Be by log with reserve ₁₀Diluent is seeded to the every dilution factor of 2-4 egg and measures.Egg was cultivated 40-48 hour, and collected the 1ml allantoic fluid from each egg.EID ₅₀For making 1/2 egg, it is negative as calculated, and the be positive dilution factor of viral infection of 1/2 egg.

Result and discussion

After with STF2.HA1-2 (VN) (SEQ ID NO:95) immunity, induce the reaction of HA-specific IgG: STF2.HA1-2 (VN) (SEQ ID NO:95) exempt from different originality be by, in the 0th and 14 day dosage range, test through subcutaneous immune BALB/c mouse (15/ group) with 10,3 and 1 μ g protein one.The negative control group mice is to cause with TBS to exempt from or exempt from without causing.Rose (the 21st day) back 7 days in short, by the reaction of ELISA check HA-specific IgG.The result proves, causes and exempts from the STF2.HA1-2 (VN) (SEQ ID NO:95) of 10,3 or 1 μ g, can induce unanimity and significant HA-specific IgG reaction (Figure 14-16) by dose-dependent manner.

Causing hands-free confession with STF2.HA1-2 (VN) (SEQ ID NO:95) avoids exciting with the deadly of influenza A Protective effect: aforesaid serological analysis proves that mice causes and exempts from STF2.HA1-2 (VN) (SEQ ID NO:95), can produce the antibody response of the natural HA of identification.Be the assessment effect, satisfy same mouse was given 10LD in the 28th day through the intranasal throwing ₉₀(6x10 ³EID) A/ Vietnam/1203/04 virus and exciting.Reach 21 days in the survival and the clinical sign that excite the back to investigate mice every day.As shown in figure 17, through TBS-cause exempt from or without any mice immunized at early as after exciting five days, promptly present and infect symptom (body weight runs off and lower clinical scores), and all mices are in exciting back the 9th day all dead.For causing the mice of exempting from, clear and definite relation is arranged between institute's acceptable dose and effect through STF2.HA1-2 (VN).Cause the mice of exempting from through the STF2.HA1-2 of 10 μ g (VN) (SEQ ID NO:95), prove remarkable enhanced protective effect.Cause the mice of exempting from through the STF2.HA1-2 of 3 or 1 μ g (VN) (SEQ ID NO:95), proving for effect has appropriateness respectively to insignificant impact.Clinical parameter is same relevant with dosage, and the animal of accepting maximum dose level STF2.HA1-2 (VN) (SEQ ID NO:95) presents the gentleest disease symptom.These results prove that through the STF2.HA1-2 of escherichia coli expression (VN) (SEQ ID NO:95), inducing can be in vivo successfully protecting BALB/c mouse to avoid the deadly HA-specific immune response that excites of toxicity A type influenza virus.

SEQ ID NO:1 A/ Puerto Rico/8/34HA

MKANLLVLLSALAAADADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDSHNGKLCRLKGI

APLQLGKCNIAGWLLGNPECDPLLPVRSWSYIVETPNSENGICYPGDFIDYEELREQLSSVSSFERF

EIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTEKEGSYPKLKNSYVNKKGKEVLVLWGIH

HPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQAGRMNYYWTLLKPGDTIIFEAN

GNLIAPMYAFALSRGFGSGIITSNASMHECNTKCQTPLGAINSSLPYQNIHPVTIGECPKYVRSAKL

RMVTGLRNIPSIQSRGLFGAIAGFIEGGWTGMIDGWYGYHHQNEQGSGYAADQKSTQNAINGITN

KVNTVIEKMNIQFTAVGKEFNKLEKRMENLNKKVDDGFLDIWTYNAELLVLLENERTLDFHDSN

VKNLYEKVKSQLKNNAKEIGNGCFEFYHKCDNECMESVRNGTYDYPKYSEESKLNREKVDGVK

LESMGIYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI

SEQ IDNO:2 A/ Vietnam/1203/2004HA

MEKIVLLFAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKKHNGKLCDLDGVKP

LILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPVNDLCYPGDFNDYEELKHLLSRINHFEKIQIIPKSSWSSHEASLGVSSACPYQGKSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVLWGIHHP

NDAAEQTKLYQNPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFI

APEYAYKIVKKGDSTIMKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIGECPKYVKSNRLVLA

TGLRNSPQRERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDG

VTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFH

DSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESVRNGTDYPQYSEEARLKREEISGV

KLESIGIYQILSIYSTVASSLALAIMVAGLSLWMCSNGSLQCR

SEQ ID NO:3 A/ Indonesia/5/2005HA

MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKP

LILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPTNDLCYPGSFNDYEELKHLLSRINHFEKIQI

IPKSSWSDHEASSGVSSACPYLGSPSFFRNVVWLIKKNSTYPTIKKSYNNTNQEDLLVLWGIHHPN

DAAEQTRLYQNPTTYISIGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIA

PEYAYKIVKKGDSAIMKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIGECPKYVKSNRLVLAT

GLRNSPQRESRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGV

TNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHD

SNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESIRNGTYNYPQYSEEARLKREEISGV

KLESIGTYQILSIYSTVASSLALAIMMAGLSLWMCSNGSLQCRICI

SEQ ID NO:4 A/ New Caledonia/20/1999

MKAKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDSHNGKLCLLKGIA

PLQLGNCSVAGWILGNPECELLISKESWSYIVETPNPENGTCYPGYFADYEELREQLSSVSSFERFEI

FPKESSWPNHTVTGVSASCSHNGKSSFYRNLLWLTGKNGLYPNLSKSYVNNKEKEVLVLWGVHH

PPNIGNQRALYHTENAYVSVVSSHYSRRFTPEIAKRPKVRDQEGRINYYWTLLEPGDTIIFEANGN

LIAPWYAFALSRGFGSGIITSNAPMDECDAKCQTPQGAINSSLPFQNVHPVTIGECPKYVRSAKLR

MVTGLRNIPSIQSRGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITN

KVNSVIEKMNTQFTAVGKEFNKLERRMENLNKKVDDGFLDIWTYNAELLVLLENERTLDFHDSN

VKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNNECMESVKNGTYDYPKYSEESKLNREKIDGVKL

ESMGVYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI

SEQ ID NO:5 A/ south Caro comes that/1/18HA

MEARLLVLLCAFAATNADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDSHNGKLCKLKGIA

PLQLGKCNIAGWLLGNPECDLLLTASSWSYIVETSNSENGTCYPGDFIDYEELREQLSSVSSFEKFE

IFPKTSSWPNHETTKGVTAACSYAGASSFYRNLLWLTKKGSSYPKLSKSYVNNKGKEVLVLWGV

HHPPTGTDQQSLYQNADAYVSVGSSKYNRRFTPEIAARPKVRDQAGRMNYYWTLLEPGDTITFE

ATGNLIAPWYAFALNRGSGSGIITSDAPVHDCNTKCQTPHGAINSSLPFQNIHPVTIGECPKYVRST

KLRMATGLRNIPSIQSRGLFGAIAGFIEGGWTGMIDGWYGYHHQNEQGSGYAADQKSTQNAIDGI

TNKVNSVIEKMNTQFTAVGKEFNNLERRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDFHDS

NVRNLYEKVKSQLKNNAKEIGNGCFEFYHKCDDACMESVRNGTYDYPKYSEESKLNREEIDGVK

LESMGVYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI

SEQ ID NO:6 A/ winconsin/67/2005HA

QKLPGNDNSTATLCLGHHAVPNGTIVKTITNDQIEVTNATELVQSSSTGGICDSPHQILDGENCTLID

ALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNDESFNWTGVTQ

NGTSSACKRRSNNSFFSRLNWLTHLKFKYPALNVTMPNNEKFDKLYIWGVHHPGTDNDQIFLHA

QASGRITVSTKRSQQTVIPNIGSRPRIRNIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSI

MRSDAPIGKCNSECITPNGSIPNDKPFQNVNRITYGACPRYVKQNTLKLATGMRNVPEKQTR

SEQ ID NO:7 A/X31 hypotype H3N2HA

QDLPGNDNSTATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSSSTGKICNNPHRILDGIDCTLID

ALLGDPHCDVFQNETWDLFVERSKAFSNCYPYDVPDYASLRSLVASSGTLEFITEGFTWTGVIQNG

GSNACKRGPGSGFFSRLNWLTKSGSTYPVLNVTMPNNDNFDKLYIWGIHHPSTNQEQTSLYVQAS

GRVTVSTRRSQQTIIPNIGSRPWVRGLSSRISIYWTIVKPGDVLVINSNGNLIAPRGYFKMRTGKSSI

MRSDAPIDTCISECITPNGSIPNDKPFQNVNKITYGACPKYVKQNTLKLATGMRNVPEKQT

SEQ ID NO：8 PR/8 HA1-1

SHNGKLCRLKGIAPLQLGKCNIAGWLLGNPECDPLLPVRSWSYIVETPNSENGICYPGDFIDYEEL

REQLSSVSSFERFEIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTEKEGSYPKLKNSYVNK

KGKEVLVLWGIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQAGRMNYYW

TLLKPGDTIIFEANGNLIAPMYAFALSRGFGSGIITSNASMHECNTKCQTPLGAINSSLPYQNIHPVT

IGECPKYVR

SEQ ID NO：9 PR/8HA1-2

KGIAPLQLGKCNIAGWLLGNPECDPLLPVRSWSYIVETPNSENGICYPGDFIDYEELREQLSSVSSF

ERFEIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTEKEGSYPKLKNSYVNKKGKEVLVLW

GIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQAGRMNYYWTLLKPGDTIIF

EANGNLIAPMYAFALSRGFGSGIITS

SEQ ID NO：10 PR/8HA1-3

NSENGICYPGDFIDYEELREQLSSVSSFERFEIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWL

TEKEGSYPKLKNSYVNKKGKEVLVLWGIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAE

RPKVRDQAGRMNYYWTLLKPGDTIIFEANGNLIAPMYAFALSRG

SEQ ID NO：11 VNHA1-1

EKKHNGKLCDLDGVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPVNDLCYPGDFND

YEELKHLLSRINHFEKIQIIPKSSWSSHEASLGVSSACPYQGKSSFFRNVVWLIKKNSTYPTIKRSYN

NTNQEDLLVLWGIHHPNDAAEQTKLYQNPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFW

TILKPNDAINFESNGNFIAPEYAYKIVKKGDSTIMKSELEYGNCNTKCQTPMGAINSSMPFHNIHPL

TIGECPKYVK

SEQ ID NO：12 VN HA1-2

GVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPVNDLCYPGDFNDYEELKHLLSRINH

FEKIQIIPKSSWSSHEASLGVSSACPYQGKSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTKLYQNPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSTIMKSE

SEQ ID NO：13 VN HA1-3

NDLCYPGDFNDYEELKHLLSRINHFEKIQIIPKSSWSSHEASLGVSSACPYQGKSSFFRNVVWLIKK

NSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTKLYQNPTTYISVGTSTLNQRLVPRIATRSKV

NGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKG

SEQ ID NO：14 IND HA1-1

EKTHNGKLCDLDGVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPTNDLCYPGSFND

YEELKHLLSRINHFEKIQIIPKSSWSDHEASSGVSSACPYLGSPSFFRNVVWLIKKNSTYPTIKKSYN

NTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISIGTSTLNQRLVPKIATRSKVNGQSGRMEFFWT

ILKPNDAINFESNGNFIAPEYAYKIVKKGDSAIMKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLT

IGECPKYVK

SEQ ID NO：15 IND HA1-2

GVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPTNDLCYPGSFNDYEELKHLLSRINHF

EKIQIIPKSSWSDHEASSGVSSACPYLGSPSFFRNVVWLIKKNSTYPTIKKSYNNTNQEDLLVLWGI

HHPNDAAEQTRLYQNPTTYISIGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKPNDAINFESNG

NFIAPEYAYKIVKKGDSAIMKSE

SEQ ID NO：16 IND HA1-3

NDLCYPGSFNDYEELKHLLSRINHFEKIQIIPKSSWSDHEASSGVSSACPYLGSPSFFRNVVWLIKK

NSTYPTIKKSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISIGTSTLNQRLVPKIATRSKVN

GQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVK KG

SEQ ID NO：17 NC HA1-1

SHNGKLCLLKGIAPLQLGNCSVAGWILGNPECELLISKESWSYIVETPNPENGTCYPGYFADYEEL

REQLSSVSSFERFEIFPKESSWPNHTVTGVSASCSHNGKSSFYRNLLWLTGKNGLYPNLSKSYVNN

KEKEVLVLWGVHHPPNIGNQRALYHTENAYVSVVS SHYSRRFTPEIAKRPKVRDQEGRINYYWTL

LEPGDTIIFEANGNLIAPWYAFALSRGFGSGIITSNAPMDECDAKCQTPQGAINSSLPFQNVHPVTIG

ECPKYVR

SEQ ID NO：18 NC HA1-2

KGIAPLQLGNCSVAGWILGNPECELLISKESWSYIVETPNPENGTCYPGYFADYEELREQLSSVSSF

ERFEIFPKESSWPNHTVTGVSASCSHNGKSSFYRNLLWLTGKNGLYPNLSKSYVNNKEKEVLVLW

GVHHPPNIGNQRALYHTENAYVSVVSSHYSRRFTPEIAKRPKVRDQEGRINYYWTLLEPGDTIIFE

ANGNLIAPWYAFALSRGFGSGIITS

SEQ ID NO：19 NC HA1-3

NPENGTCYPGYFADYEELREQLSSVSSFERFEIFPKESSWPNHTVTGVSASCSHNGKSSFYRNLLW

LTGKNGLYPNLSKSYVNNKEKEVLVLWGVHHPPNIGNQRALYHTENAYVSVVSSHYSRRFTPEIA

KRPKVRDQEGRINYYWTLLEPGDTIIFEANGNLIAPWYAFALSRG

SEQ ID NO：20 WIS HA1-1

QSSSTGGICDSPHQILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASL

RSLVASSGTLEFNDESFNWTGVTQNGTSSACKRRSNNSFFSRLNWLTHLKFKYPALNVTMPNNEK

FDKLYIWGVHHPGTDNDQIFLHAQASGRITVSTKRSQQTVIPNIGSRPRIRNIPSRISIYWTIVKPGDI

LLINSTGNLIAPRGYFKIRSGKSSIMRSDAPIGKCNSECITPNGSIPNDKPFQNVNRITYGACPRYVK

SEQ ID NO：21 WIS HA1-2

SPHQILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTL

EFNDESFNWTGVTQNGTSSACKRRSNNSFFSRLNWLTHLKFKYPALNVTMPNNEKFDKLYIWGV

HHPGTDNDQIFLHAQASGRITVSTKRSQQTVIPNIGSRPRIRNIPSRISIYWTIVKPGDILLINSTGNLI

APRGYFKIRSGKSSIMRSD

SEQ ID NO：22 WIS HA1-3

SNCYPYDVPDYASLRSLVASSGTLEFNDESFNWTGVTQNGTSSACKRRSNNSFFSRLNWLTHLKF

KYPALNVTMPNNEKFDKLYIWGVHHPGTDNDQIFLHAQASGRITVSTKRSQQTVIPNIGSRPRIRNI

PSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSG

SEQ ID NO:23 A/ Puerto Rico/8/34HA0s

DTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDSHNGKLCRLKGIAPLQLGKCNIAGWLLGNP

ECDPLLPVRSWSYIVETPNSENGICYPGDFIDYEELREQLSSVSSFERFEIFPKESSWPNHNTNGVTA

ACSHEGKSSFYRNLLWLTEKEGSYPKLKNSYVNKKGKEVLVLWGIHHPPNSKEQQNLYQNENAY

VSVVTSNYNRRFTPEIAERPKVRDQAGRMNYYWTLLKPGDTIIFEANGNLIAPMYAFALSRGFGS

GIITSNASMHECNTKCQTPLGAINSSLPYQNIHPVTIGECPKYVRSAKLRMVTGLRNIPSIQSRGLF

GAIAGFIEGGWTGMIDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNTVIEKMNIQFTAVG

KEFNKLEKRMENLNKKVDDGFLDIWTYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNA

KEIGNGCFEFYHKCDNECMESVRNGTYDYPKYSEESKLNREKVDGVKLESMGIYQ

SEQ ID NO：24 PR/8HA1-2mut

KGAAPLQLGKCNIAGWLLGNPECDPLLPVRSWSDIAETPNSENGICYPGDFIDYEELREQLSSVSS

FERFEIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTEKEGSYPKLKNSYVNKKGKEVLVL

WGIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQAGRMNYYWTLLKPGDTI

IFEANGNLIAPMYAFALSRGFGSGIITS

SEQ ID NO：25 PR/8HA1-3mut

NSENEICYPGDFIDKEELREQLSSVSSFERFEIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWL

TEKEGSYPKLKNSYVNKKGKEVLVLWGIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAE

RPKVRDQAGRMNYYWTLLKPGDTIIFEANGNLIAPMYAAALSRG

SEQ ID NO：26 VN HA1-2mut

GAKPLSLRDCSVAGWLLGNPMCDEFINVPEWSDIAEKANPVNDLCYPGDFNDYEELKHLLSRINH

FEKIQIIPKSSWSSHEASLGVSSACPYQGKSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVLWG

IHHPNDAAEQTKLYQNPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINFESN

GNFIAPEYAYKIVKKGDSTIMKSE

SEQ ID NO：27 VN HA1-3mut

NDLCYPGDFNDKEELKHLLSRINHFEKIQIIPKSSWSSHEASLGVSSACPYQGKSSFFRNVVWLIKK

NSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTKLYQNPTTYISVGTSTLNQRLVPRIATRSKV

NGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIAKKG

SEQ ID NO：28 IND HA1-2mut

GAKPLSLRDCSVAGWLLGNPMCDEFINVPEWSDIAEKANPTNDLCYPGSFNDYEELKHLLSRINH

FEKIQIIPKSSWSDHEASSGVSSACPYLGSPSFFRNVVWLIKKNSTYPTIKKSYNNTNQEDLLVLWG

IHHPNDAAEQTRLYQNPTTYISIGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKPNDAINFESN

GNFIAPEYAYKIVKKGDSAIMKSE

SEQ ID NO：29 IND HA1-3mut

NDLCYPGSFNDKEELKHLLSRINHFEKIQIIPKSSWSDHEASSGVSSACPYLGSPSFFRNVVWLIKK

NSTYPTIKKSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISIGTSTLNQRLVPKIATRSKVN

GQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIAKKG

SEQ ID NO：30 NC HA1-2mut

KGAAPLQLGNCSVAGWILGNPECELLISKESWSDIAETPNPENGTCYPGYFADYEELREQLSSVSS

FERFEIFPKESSWPNHTVTGVSASCSHNGKSSFYRNLLWLTGKNGLYPNLSKSYVNNKEKEVLVL

WGVHHPPNIGNQRALYHTENAYVSVVSSHYSRRFTPEIAKRPKVRDQEGRINYYWTLLEPGDTIIF

EANGNLIAPWYAFALSRGFGSGIITS

SEQ ID NO：31 NC HA1-3mut

NPENETCYPGYFADKEELREQLSSVSSFERFEIFPKESSWPNHTVTGVSASCSHNGKSSFYRNLLW

LTGKNGLYPNLSKSYVNNKEKEVLVLWGVHHPPNIGNQRALYHTENAYVSVVSSHYSRRFTPEIA

KRPKVRDQEGRINYYWTLLEPGDTIIFEANGNLIAPWYAAALSRG

SEQ ID NO：32 WIS HA1-2mut

SPHQALDGENCTLIDALLGDPQCDGFQNKKWDDFAERSKAYSNCYPYDVPDYASLRSLVASSGTL

EFNDESFNWTGVTQNGTSSACKRRSNNSFFSRLNWLTHLKFKYPALNVTMPNNEKFDKLYIWGV

HHPGTDNDQIFLHAQASGRITVSTKRSQQTVIPNIGSRPRIRNIPSRISIYWTIVKPGDILLINSTGNLI

APRGYFKIRSGKSSIMRSD

SEQ ID NO：33 WIS HA1-3mut

SNCYPKDSPDEASLRSLVASSGTDEFNDESFNWTGVTQNGTSSACKRRSNNSFFSRLNWLTHLKF

KYPALNVTMPNNEKFDKLYIWGVHHPGTDNDQIFLHAQASGRITVSTKRSQQTVIPNIGSRPRIRNI

PSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSG

SEQ ID NO:34 A/ pig/Hong Kong/9/98HA (numbering BAB85618)

DKICIGYQSTNSTETVDTLTETNVPVTHAKELLHTEHNGMLCATNLGHPLILDTCTIEGLIYGNPSC

DLLLGGREWSYIVERPSAVNGMCYPGNVENLEELRSLFSSASSYQRIQIFPDTIWNVSYSGTSKAC

SDSFYRSMRWLTQKNNAYPIQDAQYTNNRGKSILFMWGINHPPTDTVQTNLYTRTDTTTSVTTED

INRTFKPVIGPRPLVNGLHGRIDYYWSVLKPGQTLRVRSNGNLIAPWYGHILSGESHGRILKTDLNS

GNCVVQCQTERGGLNTTLPFHNVSKYAFGNCPKYVGVKSLKLAVGLRNVPARS SR

SEQ ID NO:35 A/ likes to know/2/1968HA (numbering BAF37221)

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSSSTGKIC

NNPHRILDGIDCTLIDALLGDPHCDVFQNETWDLFVERSKAFSNCYPYDVPDYASLRSLVASSGTL

EFITEGFTWTGVTQNGGSNACKRGPGSGFFSRLNWLTKSGSTYPVLNVTMPNNDNFDKLYIWGIH

HPSTNQEQTSLYVQASGRVTVSTRRSQQTIIPNIGSRPWVRGLSSRISIYWTIVKPGDVLVINSNGNL

IAPRGYFKMRTGKSSIMRSDAPIDTCISECITPNGSIPNDKPFQNVNKITYGACPKYVKQNTLKLAT

GMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQINGKLN

RVIEKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFE

KTRRQLRENAEEMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD

WILWISFAISCFLLCVVLLGFIMWACQRGNIRCNICI

SEQ ID NO:36 Lee B//40HA (numbering NP_056660)

MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTQTRG

KLCPNCFNCTDLDVALGRPKCMGNTPSAKVSILHEVKPATSGCFPIMHDRTKIRQLPNLLRGYENI

RLSTSNVINTETAPGGPYKVGTSGSCPNVANGNGFFNTMAWVIPKDNNKTAINPVTVEVPYICSEG

EDQITVWGFHSDDKTQMERLYGDSNPQKFTSSANGVTTHYVSQIGGFPNQTEDEGLKQSGRIVV

DYMVQKPGKTGTIVYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTG

EHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWHGYTSHGA

HGVAVAADLKSTQEAINKITKNLNYLSELEVKNLQRLSGAMNELHDEILELDEKVDDLRADTISSQ

IELAVLLSNEGIINSEDEHLLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFNAGD

FSLPTFDSLNITAASLNDDGLDNHTILLYYSTAASSLAVTLMIAIFIVYMVSRDNVSCSICL

SEQ ID NO:37 C/ Johannesburg/1/66 (numbering AAW73083)

MFFSLLLVLGLTEAEKIKICLQKQVNSSFSLHNGFGGNLYATEEKRMFELVKPKAGASVLNQSTWI

GFGDSRTDKSNSAFPRSADVSAKTADKFRSLSGGSLMLSMFGPPGKVDYLYQGCGKHKVFYEGV

NWSPHAAINCYRKNWTDIKLNFQKNIYELASQSHCMSLVNALDKTIPLQVTAGTAGNCNNSFLK

NPALYTQEVKPSENKCGKENLAFFTLPTQFGTYECKLHLVASCYFIYDSKEVYNKRGCDNYFQVI

YDSSGKVVGGLDNRVSPYTGNSGDTPTMQCDMLQLKPGRYSVRSSPRFLLMPERSYCFDMKEKG

PVTAVQSIWGKGRESDYAVDQACLSTPGCMLIQKQKPYIGEADDHHGDQEMRELLSGLDYEARCI

SQSGWVNETSPFTEKYLLPPKFGRCPLAAKEESIPKIPDGLLIPTSGTDTTVTKPKSRIFGIDDLIIGL

LFVAIVEAGIGGYLLGSRKESGGGVTKESAEKGFEKIGNDIQILKSSINIAIEKLNDRISHDEQAIRDL

TLEIENARSEALLGELGIIRALLVGNISIGLQESLWELASEITNRAGDLAVEVSPGCWIIDNNICDQSC

QNFIFKFNETAPVPTIPPLDTKIDLQSDPFYWGSSLGLAITATISLAALVISGIAIC RTK

SEQ ID NO:38 Lee B//40HA1-1

TTTPTKSHFANLKGTQTRGKLCPNCFNCTDLDVALGRPKCMGNTPSAKVSILHEVKPATSGCFPIM

HDRTKIRQLPNLLRGYENIRLSTSNVINTETAPGGPYKVGTSGSCPNVANGNGFFNTMAWVIPKDN

NKTAINPVTVEVPYICSEGEDQITVWGFHSDDKTQMERLYGDSNPQKFTSSANGVTTHYVSQIGG

FPNQTEDEGLKQSGRIVVDYMVQKPGKTGTIVYQRGILLPQKVWCASGRSKVIKGSLPLIGEADC

LHEKYGGLNKSKPYYTGEHAKAIGNCPIWVK

SEQ ID NO:39 Lee B//40HA1-2

KGTQTRGKLCPNCFNCTDLDVALGRPKCMGNTPSAKVSILHEVKPATSGCFPIMHDRTKIRQLPNL

LRGYENIRLSTSNVINTETAPGGPYKVGTSGSCPNVANGNGFFNTMAWVIPKDNNKTAINPVTVE

VPYICSEGEDQITVWGFHSDDKTQMERLYGDSNPQKFTSSANGVTTHYVSQIGGFPNQTEDEGLK

QSGRIVVDYMVQKPGKTGTIVYQRGILLPQKVWCASGRSKVIKG

SEQ ID NO:40 A/ likes to know/2/68HA1-1

QSSSTGKICNNPHRILDGIDCTLIDALLGDPHCDVFQNETWDLFVERSKAFSNCYPYDVPDYASLR

SLVASSGTLEFITEGFTWTGVTQNGGSNACKRGPGSGFFSRLNWLTKSGSTYPVLNVTMPNNDNF

DKLYIWGIHHPSTNQEQTSLYVQASGRVTVSTRRSQQTIIPNIGSRPWVRGLSSRISIYWTIVKPGDV

LVINSNGNLIAPRGYFKMRTGKSSIMRSDAPIDTCISECITPNGSIPNDKPFQNVNKITYGACPKYVK

SEQ ID NO:41 A/ likes to know/2/68HA1-2

HRILDGIDCTLIDALLGDPHCDVFQNETWDLFVERSKAFSNCYPYDVPDYASLRSLVASSGTLEFIT

EGFTWTGVTQNGGSNACKRGPGSGFFSRLNWLTKSGSTYPVLNVTMPNNDNFDKLYIWGIHHPS

TNQEQTSLYVQASGRVTVSTRRSQQTIIPNIGSRPWVRGLSSRISIYWTIVKPGDVLVINSNGNLIAP

RGYFKMRTGKSSIMRSD

SEQ ID NO:42 B/ Malaysia/2506/2004HA (numbering ISDN126672)

IVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPK

CLNCTDLDVALGRPKCTGNIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHN

VINAENAPGGSYKIGTSGSCPNVTNGNGFFATMAWAVPKNDNNKTATNSLTIEVPYICTEGEDQIT

VWGFHSDNEAQMAKLYGDSKPQKFTSSANGVTTHYVSQIGGFPNQTEDGGLPQSGRIVVDYMV

QKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKER

SEQ ID NO:43 B/ Ohio/1/2005HA (numbering ISDN133312)

DRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTKTRGKLCPKCLNCTDLDVA

LGRPKCTGNIPSAEVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAEKAPGG

PYKIGTSGSCPNVTNGNGFFATMAWAVPKNDNNKTATNSLTIEVPYICTEGEDQITIWGFHSDSETQ

MAKLYGDSKPQKFTSSANGVTTHYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQ

RGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPL

KLANGTKYRPPAKLLKERGF

SEQ ID NO:44 B/ Shanghai/361/2002HA (numbering ISDN80784)

DRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPIKSHFANLKGTRTRGKLCPDCLNCTDLDVAL

GRPMCVGTTPSAKASILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYENIRLSTQNVIDAEKALGG

PYRLGTSGSCPNATSKSGFFATMAWAVPKDNNKNATNPLTVEVPYICTEGEDQITVWGFHSDDKT

QMKNLYGDSNPQKFTSSANGVTTHYVSQIGGFPDQTEDGGLPQSGRIVVDYMVQKPGKTGTIVY

QRGVLLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGHCPIWVKT

PLKLANGTKYRP

SEQ ID NO:45 B/ Malaysia/2506/2004HA1-1

TTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGNIPSARVSILHEVRPVTSGCFPIMH

DRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGSYKIGTSGSCPNVTNGNGFFATMAWAVPKNDN

NKTATNSLTIEVPYICTEGEDQITVWGFHSDNEAQMAKLYGDSKPQKFTSSANGVTTHYVSQIGGF

PNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCL

HEKYGGLNKSKPYYTGEHAKAIGNCPIWVK

SEQ ID NO:46 B/ Malaysia/2506/2004HA1-2

KGTETRGKLCPKCLNCTDLDVALGRPKCTGNIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLL

RGYEHIRLSTHNVINAENAPGGSYKIGTSGSCPNVTNGNGFFATMAWAVPKNDNNKTATNSLTIEV

PYICTEGEDQITVWGFHSDNEAQMAKLYGDSKPQKFTSSANGVTTHYVSQIGGFPNQTEDGGLPQ

SGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKG

SEQ ID NO:47 B/ Ohio/1/2005HA1-1

TTTPTKSHFANLKGTKTRGKLCPKCLNCTDLDVALGRPKCTGNIPSAEVSILHEVRPVTSGCFPIM

HDRTKIRQLPNLLRGYEHIRLSTHNVINAEKAPGGPYKIGTSGSCPNVTNGNGFFATMAWAVPKND

NNKTATNSLTIEVPYICTEGEDQITIWGFHSDSETQMAKLYGDSKPQKFTSSANGVTTHYVSQIGGF

PNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCL

HEKYGGLNKSKPYYTGEHAKAIGNCPIWVK

SEQ ID NO:48 B/ Ohio/1/2005HA1-2

KGTKTRGKLCPKCLNCTDLDVALGRPKCTGNIPSAEVSILHEVRPVTSGCFPIMHDRTKIRQLPNL

LRGYEHIRLSTHNVINAEKAPGGPYKIGTSGSCPNVTNGNGFFATMAWAVPKNDNNKTATNSLTIE

VPYICTEGEDQITIWGFHSDSETQMAKLYGDSKPQKFTSSANGVTTHYVSQIGGFPNQTEDGGLPQ

SGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKG

SEQ ID NO:49 B/ Shanghai/361/2002HA1-1

TTTPIKSHFANLKGTRTRGKLCPDCLNCTDLDVALGRPMCVGTTPSAKASILHEVRPVTSGCFPIM

HDRTKIRQLPNLLRGYENIRLSTQNVIDAEKALGGPYRLGTSGSCPNATSKSGFFATMAWAVPKDN

NKNATNPLTVEVPYICTEGEDQITVWGFHSDDKTQMKNLYGDSNPQKFTSSANGVTTHYVSQIGG

FPDQTEDGGLPQSGRIVVDYMVQKPGKTGTIVYQRGVLLPQKVWCASGRSKVIKGSLPLIGEADC

LHEKYGGLNKSKPYYTGEHAKAIGHCPIWVK

SEQ ID NO:50 B/ Shanghai/361/2002HA1-2

KGTRTRGKLCPDCLNCTDLDVALGRPMCVGTTPSAKASILHEVRPVTSGCFPIMHDRTKIRQLPNL

LRGYENIRLSTQNVIDAEKALGGPYRLGTSGSCPNATSKSGFFATMAWAVPKDNNKNATNPLTVE

VPYICTEGEDQITVWGFHSDDKTQMKNLYGDSNPQKFTSSANGVTTHYVSQIGGFPDQTEDGGLP

QSGRIVVDYMVQKPGKTGTIVYQRGVLLPQKVWCASGRSKVIKG

SEQ ID NO：51 STF2.blp

ATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAA

TCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGC

GCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGT

CTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGC

GCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCT

AACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTG

AACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAG

GACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTG

AAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCAGAAAGCGTATGAT

GTGAAAGATACAGCAGTAACAACGAAAGCTTATGCCAATAATGGTACTACACTGGATGTA

TCGGGTCTTGATGATGCAGCTATTAAAGCGGCTACGGGTGGTACGAATGGTACGGCTTCT

GTAACCGGTGGTGCGGTTAAATTTGACGCAGATAATAACAAGTACTTTGTTACTATTGGT

GGCTTTACTGGTGCTGATGCCGCCAAAAATGGCGATTATGAAGTTAACGTTGCTACTGAC

GGTACAGTAACCCTTGCGGCTGGCGCAACTAAAACCACAATGCCTGCTGGTGCGACAACT

AAAACAGAAGTACAGGAGTTAAAAGATACACCGGCAGTTGTTTCAGCAGATGCTAAAAAT

GCCTTAATTGCTGGCGGCGTTGACGCTACCGATGCTAATGGCGCTGAGTTGGTCAAAATG

TCTTATACCGATAAAAATGGTAAGACAATTGAAGGCGGTTATGCGCTTAAAGCTGGCGAT

AAGTATTACGCCGCAGATTACGATGAAGCGACAGGAGCAATTAAAGCTAAAACTACAAGT

TATACTGCTGCTGACGGCACTACCAAAACAGCGGCTAACCAACTGGGTGGCGTAGACGGT

AAAACCGAAGTCGTTACTATCGACGGTAAAACCTACAATGCCAGCAAAGCCGCTGGTCAT

GATTTCAAAGCACAACCAGAGCTGGCGGAAGCAGCCGCTAAAACCACCGAAAACCCGCTG

CAGAAAATTGATGCCGCGCTGGCGCAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTA

CAAAACCGTTTCAACTCTGCTATCACCAACCTGGGCAATACCGTAAACAATCTGTCTGAA

GCGCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCG

CAGATTCTGCAGCAGGCCGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTCCCGCAGAAC

GTGCTGAGCCTGTTACGT

SEQ ID NO：52 STF2.SG

ATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAA

TCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGC

GCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGT

CTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGC

GCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCT

AACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTG

AACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAG

GACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTG

AAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCAGAAAGCGTATGAT

GTGAAAGATACAGCAGTAACAACGAAAGCTTATGCCAATAATGGTACTACACTGGATGTA

TCGGGTCTTGATGATGCAGCTATTAAAGCGGCTACGGGTGGTACGAATGGTACGGCTTCT

GTAACCGGTGGTGCGGTTAAATTTGACGCAGATAATAACAAGTACTTTGTTACTATTGGT

GGCTTTACTGGTGCTGATGCCGCCAAAAATGGCGATTATGAAGTTAACGTTGCTACTGAC

GGTACAGTAACCCTTGCGGCTGGCGCAACTAAAACCACAATGCCTGCTGGTGCGACAACT

AAAACAGAAGTACAGGAGTTAAAAGATACACCGGCAGTTGTTTCAGCAGATGCTAAAAAT

GCCTTAATTGCTGGCGGCGTTGACGCTACCGATGCTAATGGCGCTGAGTTGGTCAAAATG

TCTTATACCGATAAAAATGGTAAGACAATTGAAGGCGGTTATGCGCTTAAAGCTGGCGAT

AAGTATTACGCCGCAGATTACGATGAAGCGACAGGAGCAATTAAAGCTAAAACTACAAGT

TATACTGCTGCTGACGGCACTACCAAAACAGCGGCTAACCAACTGGGTGGCGTAGACGGT

AAAACCGAAGTCGTTACTATCGACGGTAAAACCTACAATGCCAGCAAAGCCGCTGGTCAT

GATTTCAAAGCACAACCAGAGCTGGCGGAAGCAGCCGCTAAAACCACCGAAAACCCGCTG

CAGAAAATTGATGCCGCGCTGGCGCAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTA

CAAAACCGTTTCAACTCTGCTATCACCAACCTGGGCAATACCGTAAACAATCTGTCTGAA

GCGCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCG

CAGATTCTGCAGCAGGCCGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTCCCGCAGAAC

GTGCTGTCTCTGTTACGTAGCGGCAGTGGTAGCGGATCC

SEQ ID NO：53 STF2.HA1-1PR8

ATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAA

TCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGC

GCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGT

CTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGC

GCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCT

AACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTG

AACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAG

GACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTG

AAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCAGAAAGCGTATGAT

GTGAAAGATACAGCAGTAACAACGAAAGCTTATGCCAATAATGGTACTACACTGGATGTA

TCGGGTCTTGATGATGCAGCTATTAAAGCGGCTACGGGTGGTACGAATGGTACGGCTTCT

GTAACCGGTGGTGCGGTTAAATTTGACGCAGATAATAACAAGTACTTTGTTACTATTGGT

GGCTTTACTGGTGCTGATGCCGCCAAAAATGGCGATTATGAAGTTAACGTTGCTACTGAC

GGTACAGTAACCCTTGCGGCTGGCGCAACTAAAACCACAATGCCTGCTGGTGCGACAACT

AAAACAGAAGTACAGGAGTTAAAAGATACACCGGCAGTTGTTTCAGCAGATGCTAAAAAT

GCCTTAATTGCTGGCGGCGTTGACGCTACCGATGCTAATGGCGCTGAGTTGGTCAAAATG

TCTTATACCGATAAAAATGGTAAGACAATTGAAGGCGGTTATGCGCTTAAAGCTGGCGAT

AAGTATTACGCCGCAGATTACGATGAAGCGACAGGAGCAATTAAAGCTAAAACCACAAGT

TATACTGCTGCTGACGGCACTACCAAAACAGCGGCTAACCAACTGGGTGGCGTAGACGGT

AAAACCGAAGTCGTTACTATCGACGGTAAAACCTACAATGCCAGCAAAGCCGCTGGTCAT

GATTTCAAAGCACAACCAGAGCTGGCGGAAGCAGCCGCTAAAACCACCGAAAACCCGCTG

CAGAAAATTGATGCCGCGCTGGCGCAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTA

CAAAACCGTTTCAACTCTGCTATCACCAACCTGGGCAATACCGTAAACAATCTGTCTGAA

GCGCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCG

CAGATTTTGCAGCAGGCCGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTCCCGCAGAAC

GTGCTGTCTCTGTTACGTAGCGGCAGTGGTAGCGGATCCTCTCATAACGGTAAACTGTGT

CGTCTGAAAGGTATTGCACCACTGCAGCTGGGCAAATGCAACATCGCGGGTTGGCTGCTG

GGTAACCCTGAATGTGACCCGCTGCTGCCGGTTCGTTCCTGGAGCTACATTGTTGAAACC

CCGAACTCCGAAAACGGTATCTGCTACCCGGGCGACTTTATTGACTATGAAGAACTGCGT

GAGCAGCTGTCTTCCGTGAGCAGCTTTGAACGCTTCGAAATCTTCCCGAAGGAAAGCTCC

TGGCCGAACCACAACACTAACGGCGTGACGGCGGCTTGCTCCCACGAAGGCAAATCTTCC

TTTTATCGTAACCTGCTGTGGCTGACTGAAAAGGAAGGTTCCTACCCAAAACTGAAAAAC

AGCTATGTTAACAAAAAGGGTAAAGAAGTCCTGGTGCTGTGGGGCATCCACCACCCGCCG

AACTCCAAGGAACAGCAGAATCTGTATCAGAACGAAAACGCATACGTTTCTGTCGTTACT

TCCAACTATAACCGTCGTTTCACTCCGGAAATCGCGGAACGTCCGAAAGTACGCGACCAG

GCTGGCCGTATGAACTACTACTGGACCCTGCTGAAACCGGGTGACACTATTATCTTCGAA

GCTAACGGTAACCTGATCGCACCAATGTACGCTTTCGCACTGTCTCGTGGTTTCGGTTCC

GGCATTATCACCAGCAACGCGTCTATGCACGAATGCAACACGAAATGTCAGACGCCGCTG

GGCGCAATTAATAGCAGCCTGCCGTACCAGAACATCCACCCGGTGACTATCGGCGAATGC

CCGAAGTATGTTCGTTAATAG

SEQ ID NO：54 STF2.HA1-1.his PR8

ATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAA

TCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGC

GCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGT

CTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGC

GCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCT

AACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTG

AACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAG

GACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTG

AAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCAGAAAGCGTATGAT

GTGAAAGATACAGCAGTAACAACGAAAGCTTATGCCAATAATGGTACTACACTGGATGTA

TCGGGTCTTGATGATGCAGCTATTAAAGCGGCTACGGGTGGTACGAATGGTACGGCTTCT

GTAACCGGTGGTGCGGTTAAATTTGACGCAGATAATAACAAGTACTTTGTTACTATTGGT

GGCTTTACTGGTGCTGATGCCGCCAAAAATGGCGATTATGAAGTTAACGTTGCTACTGAC

GGTACAGTAACCCTTGCGGCTGGCGCAACTAAAACCACAATGCCTGCTGGTGCGACAACT

AAAACAGAAGTACAGGAGTTAAAAGATACACCGGCAGTTGTTTCAGCAGATGCTAAAAAT

GCCTTAATTGCTGGCGGCGTTGACGCTACCGATGCTAATGGCGCTGAGTTGGTCAAAATG

TCTTATACCGATAAAAATGGTAAGACAATTGAAGGCGGTTATGCGCTTAAAGCTGGCGAT

AAGTATTACGCCGCAGATTACGATGAAGCGACAGGAGCAATTAAAGCTAAAACCACAAGT

TATACTGCTGCTGACGGCACTACCAAAACAGCGGCTAACCAACTGGGTGGCGTAGACGGT

AAAACCGAAGTCGTTACTATCGACGGTAAAACCTACAATGCCAGCAAAGCCGCTGGTCAT

GATTTCAAAGCACAACCAGAGCTGGCGGAAGCAGCCGCTAAAACCACCGAAAACCCGCTG

CAAAAAATTGATGCCGCGCTGGCGCAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTA

CAAAACCGTTTCAACTCTGCTATCACCAACCTGGGCAATACCGTAAACAATCTGTCTGAA

GCGCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCG

CAGATTTTGCAGCAGGCCGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTCCCGCAGAAC

GTGCTGTCTCTGTTACGTAGCGGCAGTGGTAGCGGATCCTCTCATAACGGTAAACTGTGT

CGTCTGAAAGGTATTGCACCACTGCAGCTGGGCAAATGCAACATCGCGGGTTGGCTGCTG

GGTAACCCTGAATGTGACCCGCTGCTGCCGGTTCGTTCCTGGAGCTACATTGTTGAAACC

CCGAACTCCGAAAACGGTATCTGCTACCCGGGCGACTTTATTGACTATGAAGAACTGCGT

GAGCAGCTGTCTTCCGTGAGCAGCTTTGAACGCTTCGAAATCTTCCCGAAGGAAAGCTCC

TGGCCGAACCACAACACTAACGGCGTGACGGCGGCTTGCTCCCACGAAGGCAAATCTTCC

TTTTATCGTAACCTGCTGTGGCTGACTGAAAAGGAAGGTTCCTACCCAAAACTGAAAAAC

AGCTATGTTAACAAAAAGGGTAAAGAAGTCCTGGTGCTGTGGGGCATCCACCACCCGCCG

AACTCCAAGGAACAGCAGAATCTGTATCAGAACGAAAACGCATACGTTTCTGTCGTTACT

TCCAACTATAACCGTCGTTTCACTCCGGAAATCGCGGAACGTCCGAAAGTACGCGACCAG

GCTGGCCGTATGAACTACTACTGGACCCTGCTGAAACCGGGTGACACTATTATCTTCGAA

GCTAACGGTAACCTGATCGCACCAATGTACGCTTTCGCACTGTCTCGTGGTTTCGGTTCC

GGCATTATCACCAGCAACGCGTCTATGCACGAATGCAACACGAAATGTCAGACGCCGCTG

GGCGCAATTAATAGCAGCCTGCCGTACCAGAACATCCACCCGGTGACTATCGGCGAATGC

CCGAAGTATGTTCGTCATCACCATCATCACCATTAATAG

SEQ ID NO：55 STF2.HA1-2PR8

ATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAA

TCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGC

GCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGT

CTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGC

GCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCT

AACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTG

AACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAG

GACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTG

AAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCAGAAAGCGTATGAT

GTGAAAGATACAGCAGTAACAACGAAAGCTTATGCCAATAATGGTACTACACTGGATGTA

TCGGGTCTTGATGATGCAGCTATTAAAGCGGCTACGGGTGGTACGAATGGTACGGCTTCT

GTAACCGGTGGTGCGGTTAAATTTGACGCAGATAATAACAAGTACTTTGTTACTATTGGT

GGCTTTACTGGTGCTGATGCCGCCAAAAATGGCGATTATGAAGTTAACGTTGCTACTGAC

GGTACAGTAACCCTTGCGGCTGGCGCAACTAAAACCACAATGCCTGCTGGTGCGACAACT

AAAACAGAAGTACAGGAGTTAAAAGATACACCGGCAGTTGTTTCAGCAGATGCTAAAAAT

GCCTTAATTGCTGGCGGCGTTGACGCTACCGATGCTAATGGCGCTGAGTTGGTCAAAATG

TCTTATACCGATAAAAATGGTAAGACAATTGAAGGCGGTTATGCGCTTAAAGCTGGCGAT

AAGTATTACGCCGCAGATTACGATGAAGCGACAGGAGCAATTAAAGCTAAAACTACAAGT

TATACTGCTGCTGACGGCACTACCAAAACAGCGGCTAACCAACTGGGTGGCGTAGACGGT

AAAACCGAAGTCGTTACTATCGACGGTAAAACCTACAATGCCAGCAAAGCCGCTGGTCAT

GATTTCAAAGCACAACCAGAGCTGGCGGAAGCAGCCGCTAAAACCACCGAAAACCCGCTG

CAGAAAATTGATGCCGCGCTGGCGCAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTA

CAAAACCGTTTCAACTCTGCTATCACCAACCTGGGCAATACCGTAAACAATCTGTCTGAA

GCGCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCG

CAGATTCTGCAGCAGGCCGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTCCCGCAGAAC

GTGCTGTCTCTGTTACGTTCTGGTTCTGGTTCTGGTTCTAAAGGTATTGCTCCACTGCAA

CTGGGTAAATGCAATATTGCGGGTTGGCTGCTGGGCAACCCGGAATGCGATCCGCTGCTG

CCGGTCCGTTCCTGGAGCTATATTGTTGAAACTCCGAACTCTGAGAACGGCATCTGCTAT

CCAGGTGATTTCATTGACTATGAGGAACTGCGTGAACAACTGTCTTCCGTGTCTTCCTTT

GAACGTTTCGAGATTTTTCCTAAAGAATCTTCTTGGCCGAACCATAACACTAATGGTGTT

ACCGCTGCGTGCTCTCATGAAGGTAAATCTAGCTTTTACCGCAACCTGCTGTGGCTGACC

GAGAAAGAAGGTTCTTACCCGAAACTGAAAAACAGCTACGTAAACAAAAAGGGCAAGGAA

GTTCTGGTCCTGTGGGGTATCCACCATCCGCCGAACAGCAAGGAACAGCAGAATCTGTAT

CAGAACGAAAACGCATACGTATCTGTTGTTACTTCTAACTACAACCGCCGTTTCACCCCT

GAAATCGCGGAACGTCCGAAAGTGCGTGACCAGGCAGGCCGCATGAACTATTACTGGACC

CTGCTGAAGCCGGGTGATACTATCATCTTCGAAGCGAACGGTAACCTGATCGCCCCGATG

TACGCGTTCGCTCTGAGCCGTGGCTTCGGCTCTGGTATCATTACGTCTTAATAA

SEQ ID NO：56 STF2.HA1-2mut PR8

ATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAA

TCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGC

GCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGT

CTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGC

GCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCT

AACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTG

AACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAG

GACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTG

AAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCAGAAAGCGTATGAT

GTGAAAGATACAGCAGTAACAACGAAAGCTTATGCCAATAATGGTACTACACTGGATGTA

TCGGGTCTTGATGATGCAGCTATTAAAGCGGCTACGGGTGGTACGAATGGTACGGCTTCT

GTAACCGGTGGTGCGGTTAAATTTGACGCAGATAATAACAAGTACTTTGTTACTATTGGT

GGCTTTACTGGTGCTGATGCCGCCAAAAATGGCGATTATGAAGTTAACGTTGCTACTGAC

GGTACAGTAACCCTTGCGGCTGGCGCAACTAAAACCACAATGCCTGCTGGTGCGACAACT

AAAACAGAAGTACAGGAGTTAAAAGATACACCGGCAGTTGTTTCAGCAGATGCTAAAAAT

GCCTTAATTGCTGGCGGCGTTGACGCTACCGATGCTAATGGCGCTGAGTTGGTCAAAATG

TCTTATACCGATAAAAATGGTAAGACAATTGAAGGCGGTTATGCGCTTAAAGCTGGCGAT

AAGTATTACGCCGCAGATTACGATGAAGCGACAGGAGCAATTAAAGCTAAAACCACAAGT

TATACTGCTGCTGACGGCACTACCAAAACAGCGGCTAACCAACTGGGTGGCGTAGACGGT

AAAACCGAAGTCGTTACTATCGACGGTAAAACCTACAATGCCAGCAAAGCCGCTGGTCAT

GATTTCAAAGCACAACCAGAGCTGGCGGAAGCAGCCGCTAAAACCACCGAAAACCCGCTG

CAGAAAATTGATGCCGCGCTGGCGCAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTA

CAAAACCGTTTCAACTCTGCTATCACCAACCTGGGCAATACCGTAAACAATCTGTCTGAA

GCGCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCG

CAGATTTTGCAGCAGGCCGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTCCCGCAGAAC

GTGCTGTCTCTGTTAGCGAGCGGCAGTGGTAGCGGATCCAAAGGTGCGGCTCCACTGCAA

CTGGGTAAATGCAATATTGCGGGTTGGCTGCTGGGCAACCCGGAATGCGATCCGCTGCTG

CCGGTCCGTTCCTGGAGCGATATTGCGGAAACTCCGAACTCTGAGAACGGCATCTGCTAT

CCAGGTGATTTCATTGACTATGAGGAACTGCGTGAACAACTGTCTTCCGTGTCTTCCTTT

GAACGTTTCGAGATTTTTCCTAAAGAATCTTCTTGGCCGAACCATAACACTAATGGTGTT

ACCGCTGCGTGCTCTCATGAAGGTAAATCTAGCTTTTACCGCAACCTGCTGTGGCTGACC

GAGAAAGAAGGTTCTTACCCGAAACTGAAAAACAGCTACGTAAACAAAAAGGGCAAGGAA

GTTCTGGTCCTGTGGGGTATCCACCATCCGCCGAACAGCAAGGAACAGCAGAATCTGTAT

CAGAACGAAAACGCATACGTATCTGTTGTTACTTCTAACTACAACCGCCGTTTCACCCCT

GAAATCGCGGAACGTCCGAAAGTGCGTGACCAGGCAGGCCGCATGAACTATTACTGGACC

CTGCTGAAGCCGGGTGATACTATCATCTTCGAAGCGAACGGTAACCTGATCGCCCCGATG

TACGCGTTCGCTCTGAGCCGTGGCTTCGGCTCTGGTATCATTACGTCTTAATAA

SEQ ID NO：57 STF2.HA1-3 PR8

ATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAA

TCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGC

GCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGT

CTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGC

GCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCT

AACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTG

AACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAG

GACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTG

AAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCAGAAAGCGTATGAT

GTGAAAGATACAGCAGTAACAACGAAAGCTTATGCCAATAATGGTACTACACTGGATGTA

TCGGGTCTTGATGATGCAGCTATTAAAGCGGCTACGGGTGGTACGAATGGTACGGCTTCT

GTAACCGGTGGTGCGGTTAAATTTGACGCAGATAATAACAAGTACTTTGTTACTATTGGT

GGCTTTACTGGTGCTGATGCCGCCAAAAATGGCGATTATGAAGTTAACGTTGCTACTGAC

GGTACAGTAACCCTTGCGGCTGGCGCAACTAAAACCACAATGCCTGCTGGTGCGACAACT

AAAACAGAAGTACAGGAGTTAAAAGATACACCGGCAGTTGTTTCAGCAGATGCTAAAAAT

GCCTTAATTGCTGGCGGCGTTGACGCTACCGATGCTAATGGCGCTGAGTTGGTCAAAATG

TCTTATACCGATAAAAATGGTAAGACAATTGAAGGCGGTTATGCGCTTAAAGCTGGCGAT

AAGTATTACGCCGCAGATTACGATGAAGCGACAGGAGCAATTAAAGCTAAAACTACAAGT

TATACTGCTGCTGACGGCACTACCAAAACAGCGGCTAACCAACTGGGTGGCGTAGACGGT

AAAACCGAAGTCGTTACTATCGACGGTAAAACCTACAATGCCAGCAAAGCCGCTGGTCAT

GATTTCAAAGCACAACCAGAGCTGGCGGAAGCAGCCGCTAAAACCACCGAAAACCCGCTG

CAGAAAATTGATGCCGCGCTGGCGCAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTA

CAAAACCGTTTCAACTCTGCTATCACCAACCTGGGCAATACCGTAAACAATCTGTCTGAA

GCGCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCG

CAGATTCTGCAGCAGGCCGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTCCCGCAGAAC

GTGCTGTCTCTGTTACGTTCTGGTAGCGGTTCTGGCTCTAACTCTGAAAATGGTATCTGT

TACCCGGGTGATTTCATCGATTATGAGGAACTGCGTGAACAGCTGTCTAGCGTGAGCTCC

TTTGAACGCTTCGAAATCTTCCCGAAGGAATCTAGCTGGCCGAACCATAACACGAACGGT

GTGACCGCTGCTTGCTCCCACGAAGGTAAGAGCTCCTTCTACCGCAATCTGCTGTGGCTG

ACTGAAAAAGAAGGTAGCTACCCGAAACTGAAAAATTCTTACGTCAACAAGAAAGGCAAG

GAAGTGCTGGTTCTGTGGGGCATTCACCACCCACCGAACAGCAAAGAGCAACAGAACCTG

TACCAAAATGAGAACGCTTACGTTTCTGTTGTGACTTCTAACTACAATCGTCGCTTTACC

CCTGAAATCGCGGAGCGTCCAAAAGTGCGTGACCAGGCTGGTCGTATGAACTACTATTGG

ACCCTGCTGAAACCGGGCGACACCATTATCTTTGAAGCGAACGGTAACCTGATCGCGCCT

ATGTACGCGTTCGCTCTGTCTCGTGGCTAATAA

SEQ ID NO：58 STF2.HA1-3mut PR8

ATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAA

TCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGC

GCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGT

CTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGC

GCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCT

AACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTG

AACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAG

GACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTG

AAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCAGAAAGCGTATGAT

GTGAAAGATACAGCAGTAACAACGAAAGCTTATGCCAATAATGGTACTACACTGGATGTA

TCGGGTCTTGATGATGCAGCTATTAAAGCGGCTACGGGTGGTACGAATGGTACGGCTTCT

GTAACCGGTGGTGCGGTTAAATTTGACGCAGATAATAACAAGTACTTTGTTACTATTGGT

GGCTTTACTGGTGCTGATGCCGCCAAAAATGGCGATTATGAAGTTAACGTTGCTACTGAC

GGTACAGTAACCCTTGCGGCTGGCGCAACTAAAACCACAATGCCTGCTGGTGCGACAACT

AAAACAGAAGTACAGGAGTTAAAAGATACACCGGCAGTTGTTTCAGCAGATGCTAAAAAT

GCCTTAATTGCTGGCGGCGTTGACGCTACCGATGCTAATGGCGCTGAGTTGGTCAAAATG

TCTTATACCGATAAAAATGGTAAGACAATTGAAGGCGGTTATGCGCTTAAAGCTGGCGAT

AAGTATTACGCCGCAGATTACGATGAAGCGACAGGAGCAATTAAAGCTAAAACTACAAGT

TATACTGCTGCTGACGGCACTACCAAAACAGCGGCTAACCAACTGGGTGGCGTAGACGGT

AAAACCGAAGTCGTTACTATCGACGGTAAAACCTACAATGCCAGCAAAGCCGCTGGTCAT

GATTTCAAAGCACAACCAGAGCTGGCGGAAGCAGCCGCTAAAACCACCGAAAACCCGCTG

CAGAAAATTGATGCCGCGCTGGCGCAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTA

CAAAACCGTTTCAACTCTGCTATCACCAACCTGGGCAATACCGTAAACAATCTGTCTGAA

GCGCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCG

CAGATTCTGCAGCAGGCCGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTCCCGCAGAAC

GTGCTGTCTCTGTTACGTAGCGGCAGTGGTAGCGGATCCAACTCTGAAAATGAAATCTGT

TACCCGGGTGATTTCATCGATAAAGAGGAACTGCGTGAACAGCTGTCTAGCGTGAGCTCC

TTTGAACGCTTCGAAATCTTCCCGAAGGAATCTAGCTGGCCGAACCATAACACGAACGGT

GTGACCGCTGCTTGCTCCCACGAAGGTAAGAGCTCCTTCTACCGCAATCTGCTGTGGCTG

ACTGAAAAAGAAGGTAGCTACCCGAAACTGAAAAATTCTTACGTCAACAAGAAAGGCAAG

GAAGTGCTGGTTCTGTGGGGCATTCACCACCCACCGAACAGCAAAGAGCAACAGAACCTG

TACCAAAATGAGAACGCTTACGTTTCTGTTGTGACTTCTAACTACAATCGTCGCTTTACC

CCTGAAATCGCGGAGCGTCCAAAAGTGCGTGACCAGGCTGGTCGTATGAACTACTATTGG

ACCCTGCTGAAACCGGGCGACACCATTATCTTTGAAGCGAACGGTAACCTGATCGCGCCT

ATGTACGCGGCGGCTCTGTCTCGTGGCTAATAA

SEQ ID NO：59 HA1-1 PR8

ATGTCTCATAACGGTAAACTGTGTCGTCTGAAAGGTATTGCACCACTGCAGCTGGGCAAA

TGCAACATCGCGGGTTGGCTGCTGGGTAACCCTGAATGTGACCCGCTGCTGCCGGTTCGT

TCCTGGAGCTACATTGTTGAAACCCCGAACTCCGAAAACGGTATCTGCTACCCGGGCGAC

TTTATTGACTATGAAGAACTGCGTGAGCAGCTGTCTTCCGTGAGCAGCTTTGAACGCTTC

GAAATCTTCCCGAAGGAAAGCTCCTGGCCGAACCACAACACTAACGGCGTGACGGCGGCT

TGCTCCCACGAAGGCAAATCTTCCTTTTATCGTAACCTGCTGTGGCTGACTGAAAAGGAA

GGTTCCTACCCAAAACTGAAAAACAGCTATGTTAACAAAAAGGGTAAAGAAGTCCTGGTG

CTGTGGGGCATCCACCACCCGCCGAACTCCAAGGAACAGCAGAATCTGTATCAGAACGAA

AACGCATACGTTTCTGTCGTTACTTCCAACTATAACCGTCGTTTCACTCCGGAAATCGCG

GAACGTCCGAAAGTACGCGACCAGGCTGGCCGTATGAACTACTACTGGACCCTGCTGAAA

CCGGGTGACACTATTATCTTCGAAGCTAACGGTAACCTGATCGCACCAATGTACGCTTTC

GCACTGTCTCGTGGTTTCGGTTCCGGCATTATCACCAGCAACGCGTCTATGCACGAATGC

AACACGAAATGTCAGACGCCGCTGGGCGCAATTAATAGCAGCCTGCCGTACCAGAACATC

CACCCGGTGACTATCGGCGAATGCCCGAAGTATGTTCGT

SEQ ID NO：60 HA1-1.his PR8

ATGTCTCATAACGGTAAACTGTGTCGTCTGAAAGGTATTGCACCACTGCAGCTGGGCAAA

TGCAACATCGCGGGTTGGCTGCTGGGTAACCCTGAATGTGACCCGCTGCTGCCGGTTCGT

TCCTGGAGCTACATTGTTGAAACCCCGAACTCCGAAAACGGTATCTGCTACCCGGGCGAC

TTTATTGACTATGAAGAACTGCGTGAGCAGCTGTCTTCCGTGAGCAGCTTTGAACGCTTC

GAAATCTTCCCGAAGGAAAGCTCCTGGCCGAACCACAACACTAACGGCGTGACGGCGGCT

TGCTCCCACGAAGGCAAATCTTCCTTTTATCGTAACCTGCTGTGGCTGACTGAAAAGGAA

GGTTCCTACCCAAAACTGAAAAACAGCTATGTTAACAAAAAGGGTAAAGAAGTCCTGGTG

CTGTGGGGCATCCACCACCCGCCGAACTCCAAGGAACAGCAGAATCTGTATCAGAACGAA

AACGCATACGTTTCTGTCGTTACTTCCAACTATAACCGTCGTTTCACTCCGGAAATCGCG

GAACGTCCGAAAGTACGCGACCAGGCTGGCCGTATGAACTACTACTGGACCCTGCTGAAA

CCGGGTGACACTATTATCTTCGAAGCTAACGGTAACCTGATCGCACCAATGTACGCTTTC

GCACTGTCTCGTGGTTTCGGTTCCGGCATTATCACCAGCAACGCGTCTATGCACGAATGC

AACACGAAATGTCAGACGCCGCTGGGCGCAATTAATAGCAGCCTGCCGTACCAGAACATC

CACCCGGTGACTATCGGCGAATGCCCGAAGTATGTTCGTCATCACCATCATCACCAT

SEQ ID NO：61 HA1-2.his PR8

ATGAAAGGTATTGCTCCACTGCAACTGGGTAAATGCAATATTGCGGGTTGGCTGCTGGGC

AACCCGGAATGCGATCCGCTGCTGCCGGTCCGTTCCTGGAGCTATATTGTTGAAACTCCG

AACTCTGAGAACGGCATCTGCTATCCAGGTGATTTCATTGACTATGAGGAACTGCGTGAA

CAACTGTCTTCCGTGTCTTCCTTTGAACGTTTCGAGATTTTTCCTAAAGAATCTTCTTGG

CCGAACCATAACACTAATGGTGTTACCGCTGCGTGCTCTCATGAAGGTAAATCTAGCTTT

TACCGCAACCTGCTGTGGCTGACCGAGAAAGAAGGTTCTTACCCGAAACTGAAAAACAGC

TACGTAAACAAAAAGGGCAAGGAAGTTCTGGTCCTGTGGGGTATCCACCATCCGCCGAAC

AGCAAGGAACAGCAGAATCTGTATCAGAACGAAAACGCATACGTATCTGTTGTTACTTCT

AACTACAACCGCCGTTTCACCCCTGAAATCGCGGAACGTCCGAAAGTGCGTGACCAGGCA

GGCCGCATGAACTATTACTGGACCCTGCTGAAGCCGGGTGATACTATCATCTTCGAAGCG

AACGGTAACCTGATCGCCCCGATGTACGCGTTCGCTCTGAGCCGTGGCTTCGGCTCTGGT

ATCATTACGTCTCATCACCATCATCACCAT

SEQ ID NO：62 CRM.HA1-2

ATGGGTGCTGATGATGTCGTTGATTCCTCCAAAAGCTTCGTTATGGAAAATTTCTCTTCT

TATCACGGCACCAAACCGGGTTATGTGGATTCTATCCAAAAAGGCATCCAGAAACCGAAG

TCCGGTACGCAGGGTAACTATGATGACGATTGGAAAGGTTTTTACTCCACCGATAATAAA

TATGACGCGGCTGGCTACTCTGTTGACAACGAAAATCCACTGTCTGGTAAGGCTGGCGGT

GTCGTAAAGGTAACGTATCCTGGCCTGACCAAGGTGCTGGCACTGAAGGTGGATAACGCT

GAAACCATCAAAAAGGAGCTGGGCCTGAGCCTGACTGAACCGCTGATGGAGCAGGTCGGT

ACCGAGGAATTCATCAAACGCTTCGGTGATGGCGCCTCCCGTGTTGTGCTGTCCCTGCCG

TTCGCAGAAGGCTCTTCCTCTGTCGAATATATCAACAACTGGGAACAGGCTAAAGCTCTG

AGCGTCGAACTGGAAATTAACTTTGAGACCCGTGGCAAGCGTGGTCAGGACGCGATGTAC

GAATATATGGCTCAGGCTTGTGCCGGTAACCGTGTTCGCCGTTCCGTCGGTTCCTCTCTG

TCTTGCATCAACCTGGATTGGGACGTTATCCGTGATAAGACCAAAACCAAAATTGAAAGC

CTGAAGGAACACGGTCCGATCAAAAACAAAATGTCTGAATCTCCGAACAAAACCGTGTCC

GAGGAAAAAGCGAAACAGTATCTGGAAGAATTCCACCAGACTGCCCTGGAACATCCTGAA

CTGTCCGAACTGAAGACTGTAACCGGCACTAACCCGGTGTTCGCAGGCGCAAACTACGCC

GCGTGGGCGGTAAACGTTGCGCAGGTTATTGATAGCGAAACCGCAGATAACCTGGAAAAA

ACGACCGCAGCTCTGTCTATCCTGCCGGGTATCGGTTCCGTTATGGGTATTGCGGACGGC

GCTGTGCACCACAACACGGAAGAAATCGTCGCACAGTCTATCGCGCTGTCTTCTCTGATG

GTTGCTCAGGCAATTCCACTGGTAGGTGAACTGGTGGACATTGGCTTTGCGGCGTACAAC

TTCGTCGAAAGCATTATCAACCTGTTCCAGGTTGTACACAACTCTTACAACCGTCCGGCC

TACAGCCCTGGCCACAAAACCCAACCGTTTCTGCACGACGGTTATGCGGTGTCCTGGAAC

ACGGTTGAAGACTCTATCATTCGTACCGGCTTTCAGGGCGAGTCCGGCCACGATATCAAA

ATTACTGCAGAAAACACTCCGCTGCCGATCGCTGGCGTTCTGCTGCCGACCATCCCGGGT

AAGCTGGATGTGAACAAAAGCAAAACCCACATCTCTGTTAACGGTCGTAAAATTCGCATG

CGCTGTCGTGCTATCGACGGTGATGTTACCTTCTGCCGTCCGAAATCTCCAGTCTACGTG

GGCAACGGTGTTCATGCCAACCTGCACGTGGCGTTCCATCGTAGCTCTAGCGAAAAAATC

CACTCCAACGAAATCTCTAGCGATTCTATCGGTGTTCTGGGTTATCAGAAAACGGTGGAT

CATACGAAAGTCAATTCTAAACTGAGCCTGTTCTTCGAAATCAAATCTAGCGGCTCTGGA

TCCGGTTCCAAAGGCATCGCGCCGCTGCAGCTGGGTAAATGTAACATTGCGGGCTGGCTG

CTGGGTAATCCGGAATGCGATCCGCTGCTGCCGGTCCGTAGCTGGTCTTACATTGTTGAA

ACTCCGAACTCTGAGAATGGCATCTGCTACCCGGGCGATTTTATCGACTATGAAGAACTG

CGTGAACAGCTGTCTTCCGTTTCTTCCTTTGAACGTTTCGAAATCTTCCCGAAAGAAAGC

AGCTGGCCGAATCACAATACGAACGGTGTTACTGCTGCGTGTTCTCATGAAGGTAAATCC

AGCTTCTACCGTAACCTGCTGTGGCTGACCGAAAAAGAGGGTTCTTATCCTAAACTGAAA

AACAGCTACGTTAACAAAAAGGGCAAAGAAGTGCTGGTGCTGTGGGGTATCCATCACCCT

CCGAACTCTAAAGAACAACAGAATCTGTATCAGAACGAAAACGCTTACGTTTCCGTGGTG

ACCTCTAACTATAACCGTCGTTTTACCCCGGAGATTGCTGAACGTCCGAAAGTGCGCGAT

CAGGCTGGCCGTATGAACTACTATTGGACCCTGCTGAAACCGGGCGATACCATCATTTTC

GAAGCTAACGGCAACCTGATTGCTCCGATGTATGCGTTTGCTCTGTCTCGTGGCTTCGGC

TCTGGTATTATTACGTCTTAATAA

SEQ ID NO：63 LTB.HA1-2

ATGAATAAGGTTAAGTTCTACGTACTGTTTACCGCGCTGCTGTCTTCTCTGTGCGCGCAT

GGTGCTCCGCAGTCTATTACTGAACTGTGCTCTGAATACCACAACACCCAGATCTATACT

ATCAACGATAAAATCCTGAGCTATACCGAATCTATGGCAGGCAAACGCGAAATGGTTATC

ATTACCTTTAAAAGCGGCGCCACCTTTCAAGTGGAAGTTCCGGGCTCTCAGCATATTGAC

TCTCAAAAAAAAGCGATCGAACGTATGAAAGATACTCTGCGCATTACCTACCTGACCGAA

ACCAAAATCGATAAACTGTGCGTATGGAACAATAAGACTCCTAACTCTATCGCAGCTATT

TCTATGGAAAACTCTGGTAGCGGATCCGGTTCTAAAGGCATCGCGCCGCTGCAGCTGGGT

AAATGTAACATTGCGGGCTGGCTGCTGGGTAATCCGGAATGCGATCCGCTGCTGCCGGTC

CGTAGCTGGTCTTACATTGTTGAAACTCCGAACTCTGAGAATGGCATCTGCTACCCGGGC

GATTTTATCGACTATGAAGAACTGCGTGAACAGCTGTCTTCCGTTTCTTCCTTTGAACGT

TTCGAAATCTTCCCGAAAGAAAGCAGCTGGCCGAATCACAATACGAACGGTGTTACTGCT

GCGTGTTCTCATGAAGGTAAATCCAGCTTCTACCGTAACCTGCTGTGGCTGACCGAAAAA

GAGGGTTCTTATCCTAAACTGAAAAACAGCTACGTTAACAAAAAGGGCAAAGAAGTGCTG

GTGCTGTGGGGTATCCATCACCCTCCGAACTCTAAAGAACAACAGAATCTGTATCAGAAC

GAAAACGCTTACGTTTCCGTGGTGACCTCTAACTATAACCGTCGTTTTACCCCGGAGATT

GCTGAACGTCCGAAAGTGCGCGATCAGGCTGGCCGTATGAACTACTATTGGACCCTGCTG

AAACCGGGCGATACCATCATTTTCGAAGCTAACGGCAACCTGATTGCTCCGATGTATGCG

TTTGCTCTGTCTCGTGGCTTCGGCTCTGGTATTATTACGTCTTAATAA

SEQ ID NO：64 STF2.HA1-1 VN

ATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAA

TCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGC

GCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGT

CTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGC

GCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCT

AACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTG

AACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAG

GACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTG

AAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCAGAAAGCGTATGAT

GTGAAAGATACAGCAGTAACAACGAAAGCTTATGCCAATAATGGTACTACACTGGATGTA

TCGGGTCTTGATGATGCAGCTATTAAAGCGGCTACGGGTGGTACGAATGGTACGGCTTCT

GTAACCGGTGGTGCGGTTAAATTTGACGCAGATAATAACAAGTACTTTGTTACTATTGGT

GGCTTTACTGGTGCTGATGCCGCCAAAAATGGCGATTATGAAGTTAACGTTGCTACTGAC

GGTACAGTAACCCTTGCGGCTGGCGCAACTAAAACCACAATGCCTGCTGGTGCGACAACT

AAAACAGAAGTACAGGAGTTAAAAGATACACCGGCAGTTGTTTCAGCAGATGCTAAAAAT

GCCTTAATTGCTGGCGGCGTTGACGCTACCGATGCTAATGGCGCTGAGTTGGTCAAAATG

TCTTATACCGATAAAAATGGTAAGACAATTGAAGGCGGTTATGCGCTTAAAGCTGGCGAT

AAGTATTACGCCGCAGATTACGATGAAGCGACAGGAGCAATTAAAGCTAAAACCACAAGT

TATACTGCTGCTGACGGCACTACCAAAACAGCGGCTAACCAACTGGGTGGCGTAGACGGT

AAAACCGAAGTCGTTACTATCGACGGTAAAACCTACAATGCCAGCAAAGCCGCTGGTCAT

GATTTCAAAGCACAACCAGAGCTGGCGGAAGCAGCCGCTAAAACCACCGAAAACCCGCTG

CAGAAAATTGATGCCGCGCTGGCGCAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTA

CAAAACCGTTTCAACTCTGCTATCACCAACCTGGGCAATACCGTAAACAATCTGTCTGAA

GCGCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCG

CAGATTTTGCAGCAGGCCGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTCCCGCAGAAC

GTGCTGAGCCTGTTAGCGGAGAAGAAACACAATGGCAAACTGTGTGATCTGGATGGTGTG

AAACCGCTGATTCTGCGCGATTGCTCTGTGGCAGGCTGGCTGCTGGGCAACCCTATGTGT

GACGAATTCATTAACGTTCCGGAATGGTCTTACATTGTTGAAAAAGCTAACCCTGTCAAC

GATCTGTGTTACCCTGGTGACTTTAACGATTACGAAGAACTGAAGCACCTGCTGTCTCGT

ATCAATCACTTCGAGAAAATCCAGATCATCCCGAAATCCTCCTGGAGCTCCCACGAAGCT

TCTCTGGGCGTATCCTCCGCGTGCCCGTACCAGGGCAAATCCTCTTTCTTTCGTAACGTT

GTTTGGCTGATCAAGAAAAACTCCACCTACCCGACGATCAAGCGTAGCTATAATAACACC

AACCAGGAAGACCTGCTGGTTCTGTGGGGCATCCACCATCCAAACGATGCTGCGGAACAG

ACCAAGCTGTACCAGAACCCGACCACCTACATCAGCGTGGGCACCTCTACGCTGAACCAG

CGTCTGGTACCGCGTATCGCAACCCGCAGCAAGGTAAACGGTCAAAGCGGCCGCATGGAA

TTTTTCTGGACCATCCTGAAACCGAACGACGCAATCAACTTCGAATCTAACGGCAATTTC

ATCGCTCCGGAGTATGCGTACAAAATCGTAAAGAAAGGTGATAGCACTATCATGAAATCC

GAGCTGGAATATGGCAACTGTAACACCAAATGCCAGACCCCGATGGGTGCAATCAACTCC

TCCATGCCGTTTCACAACATTCACCCGCTGACTATCGGCGAATGTCCGAAATACGTTAAA

TAGTAA

SEQ ID NO:65 FOR primer

AGTGGCTGAGCCTGTTAGCGGAGAAGAAACACAATGGCAAACTGTG

SEQ ID NO:66REV primer

AGTCGCTCAGCTTACTATTTAACGTATTTCGGACATTCGCCGATAGTCAG

SEQ ID NO：67 HA0s VN

GTGCTGAGCCTGTTACGTCAGATTTGTATCGGCTACCACGCAAACAACTCTACCGAGCAA

GTTGATACCATCATGGAGAAAAACGTGACCGTTACTCACGCGCAGGACATCCTGGAGAAG

AAACACAATGGCAAACTGTGTGATCTGGATGGTGTGAAACCGCTGATTCTGCGCGATTGC

TCTGTGGCAGGCTGGCTGCTGGGCAACCCTATGTGTGACGAATTCATTAACGTTCCGGAA

TGGTCTTACATTGTTGAAAAAGCTAACCCTGTCAACGATCTGTGTTACCCTGGTGACTTT

AACGATTACGAAGAACTGAAGCACCTGCTGTCTCGTATCAATCACTTCGAGAAAATCCAG

ATCATCCCGAAATCCTCCTGGAGCTCCCACGAAGCTTCTCTGGGCGTATCCTCCGCGTGC

CCGTACCAGGGCAAATCCTCTTTCTTTCGTAACGTTGTTTGGCTGATCAAGAAAAACTCC

ACCTACCCGACGATCAAGCGTAGCTATAATAACACCAACCAGGAAGACCTGCTGGTTCTG

TGGGGCATCCACCATCCAAACGATGCTGCGGAACAGACCAAGCTGTACCAGAACCCGACC

ACCTACATCAGCGTGGGCACCTCTACGCTGAACCAGCGTCTGGTACCGCGTATCGCAACC

CGCAGCAAGGTAAACGGTCAAAGCGGCCGCATGGAATTTTTCTGGACCATCCTGAAACCG

AACGACGCAATCAACTTCGAATCTAACGGCAATTTCATCGCTCCGGAGTATGCGTACAAA

ATCGTAAAGAAAGGTGATAGCACTATCATGAAATCCGAGCTGGAATATGGCAACTGTAAC

ACCAAATGCCAGACCCCGATGGGTGCAATCAACTCCTCCATGCCGTTTCACAACATTCAC

CCGCTGACTATCGGCGAATGTCCGAAATACGTTAAATCCAATCGTCTGGTTCTGGCTACC

GGTCTGCGTAACTCCCCACAGCGTGAACGTCGTCGTAAGAAACGTGGTCTGTTTGGCGCG

ATCGCTGGTTTCATCGAGGGCGGCTGGCAGGGTATGGTTGATGGCTGGTACGGTTATCAT

CATTCCAATGAACAGGGTTCCGGCTACGCCGCAGATAAAGAAAGCACTCAGAAAGCAATT

GATGGCGTAACTAACAAAGTAAATTCTATCATTGATAAAATGAACACCCAGTTCGAGGCG

GTTGGTCGTGAGTTCAACAACCTGGAACGCCGTATCGAAAACCTGAACAAGAAAATGGAA

GACGGTTTTCTGGATGTGTGGACTTACAATGCTGAACTGCTGGTGCTGATGGAAAACGAG

CGTACCCTGGACTTCCACGACAGCAACGTCAAAAATCTGTATGACAAAGTCCGTCTGCAG

CTGCGTGATAACGCTAAAGAGCTGGGTAATGGCTGCTTCGAGTTCTATCACAAATGCGAC

AACGAATGCATGGAATCTGTCCGCAACGGCACTTACGATTATCCGCAGTACTCCGAAGAA

GCGCGCCTGAAACGCGAAGAGATCTCCGGTGTGAAGCTGGAGTCTATTGGCATCTACCAG

ATCCTGTCCATCTACAGCACCTAGTAAGCTGAGCGCCTACGCAGC

SEQ ID NO：68 STF2.HA1-2 VN

ATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAA

TCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGC

GCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGT

CTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGC

GCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCT

AACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTG

AACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAG

GACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTG

AAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCAGAAAGCGTATGAT

GTGAAAGATACAGCAGTAACAACGAAAGCTTATGCCAATAATGGTACTACACTGGATGTA

TCGGGTCTTGATGATGCAGCTATTAAAGCGGCTACGGGTGGTACGAATGGTACGGCTTCT

GTAACCGGTGGTGCGGTTAAATTTGACGCAGATAATAACAAGTACTTTGTTACTATTGGT

GGCTTTACTGGTGCTGATGCCGCCAAAAATGGCGATTATGAAGTTAACGTTGCTACTGAC

GGTACAGTAACCCTTGCGGCTGGCGCAACTAAAACCACAATGCCTGCTGGTGCGACAACT

AAAACAGAAGTACAGGAGTTAAAAGATACACCGGCAGTTGTTTCAGCAGATGCTAAAAAT

GCCTTAATTGCTGGCGGCGTTGACGCTACCGATGCTAATGGCGCTGAGTTGGTCAAAATG

TCTTATACCGATAAAAATGGTAAGACAATTGAAGGCGGTTATGCGCTTAAAGCTGGCGAT

AAGTATTACGCCGCAGATTACGATGAAGCGACAGGAGCAATTAAAGCTAAAACCACAAGT

TATACTGCTGCTGACGGCACTACCAAAACAGCGGCTAACCAACTGGGTGGCGTAGACGGT

AAAACCGAAGTCGTTACTATCGACGGTAAAACCTACAATGCCAGCAAAGCCGCTGGTCAT

GATTTCAAAGCACAACCAGAGCTGGCGGAAGCAGCCGCTAAAACCACCGAAAACCCGCTG

CAGAAAATTGATGCCGCGCTGGCGCAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTA

CAAAACCGTTTCAACTCTGCTATCACCAACCTGGGCAATACCGTAAACAATCTGTCTGAA

GCGCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCG

CAGATTTTGCAGCAGGCCGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTCCCGCAGAAC

GTGCTGAGCCTGTTAGCAGGTGTGAAACCGCTGATTCTGCGCGATTGCTCTGTGGCAGGC

TGGCTGCTGGGCAACCCTATGTGTGACGAATTCATTAACGTTCCGGAATGGTCTTACATT

GTTGAAAAAGCTAACCCTGTCAACGATCTGTGTTACCCTGGTGACTTTAACGATTACGAA

GAACTGAAGCACCTGCTGTCTCGTATCAATCACTTCGAGAAAATCCAGATCATCCCGAAA

TCCTCCTGGAGCTCCCACGAAGCTTCTCTGGGCGTATCCTCCGCGTGCCCGTACCAGGGC

AAATCCTCTTTCTTTCGTAACGTTGTTTGGCTGATCAAGAAAAACTCCACCTACCCGACG

ATCAAGCGTAGCTATAATAACACCAACCAGGAAGACCTGCTGGTTCTGTGGGGCATCCAC

CATCCAAACGATGCTGCGGAACAGACCAAGCTGTACCAGAACCCGACCACCTACATCAGC

GTGGGCACCTCTACGCTGAACCAGCGTCTGGTACCGCGTATCGCAACCCGCAGCAAGGTA

AACGGTCAAAGCGGCCGCATGGAATTTTTCTGGACCATCCTGAAACCGAACGACGCAATC

AACTTCGAATCTAACGGCAATTTCATCGCTCCGGAGTATGCGTACAAAATCGTAAAGAAA

GGTGATAGCACTATCATGAAATCCGAGTAGTAA

SEQ ID NO:69 FOR primer

AGTCGCTGAGCCTGTTAGCAGGTGTGAAACCGCTGATTCTGCGCGATTG

SEQ ID NO:70 REV primer

TGACGCTCAGCTTACTACTCGGATTTCATGATAGTGCTATCACCTTTC

SEQ ID NO：71 STF2.HA1-2mut VN

ATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAA

TCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGC

GCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGT

CTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGC

GCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCT

AACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTG

AACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAG

GACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTG

AAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCAGAAAGCGTATGAT

GTGAAAGATACAGCAGTAACAACGAAAGCTTATGCCAATAATGGTACTACACTGGATGTA

TCGGGTCTTGATGATGCAGCTATTAAAGCGGCTACGGGTGGTACGAATGGTACGGCTTCT

GTAACCGGTGGTGCGGTTAAATTTGACGCAGATAATAACAAGTACTTTGTTACTATTGGT

GGCTTTACTGGTGCTGATGCCGCCAAAAATGGCGATTATGAAGTTAACGTTGCTACTGAC

GGTACAGTAACCCTTGCGGCTGGCGCAACTAAAACCACAATGCCTGCTGGTGCGACAACT

AAAACAGAAGTACAGGAGTTAAAAGATACACCGGCAGTTGTTTCAGCAGATGCTAAAAAT

GCCTTAATTGCTGGCGGCGTTGACGCTACCGATGCTAATGGCGCTGAGTTGGTCAAAATG

TCTTATACCGATAAAAATGGTAAGACAATTGAAGGCGGTTATGCGCTTAAAGCTGGCGAT

AAGTATTACGCCGCAGATTACGATGAAGCGACAGGAGCAATTAAAGCTAAAACCACAAGT

TATACTGCTGCTGACGGCACTACCAAAACAGCGGCTAACCAACTGGGTGGCGTAGACGGT

AAAACCGAAGTCGTTACTATCGACGGTAAAACCTACAATGCCAGCAAAGCCGCTGGTCAT

GATTTCAAAGCACAACCAGAGCTGGCGGAAGCAGCCGCTAAAACCACCGAAAACCCGCTG

CAGAAAATTGATGCCGCGCTGGCGCAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTA

CAAAACCGTTTCAACTCTGCTATCACCAACCTGGGCAATACCGTAAACAATCTGTCTGAA

GCGCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCG

CAGATTTTGCAGCAGGCCGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTCCCGCAGAAC

GTGCTGAGCCTGTTAGCGGGTGCGAAACCGCTGTCTCTGCGCGATTGCTCTGTGGCAGGC

TGGCTGCTGGGCAACCCTATGTGTGACGAATTCATTAACGTTCCGGAATGGTCTGATATT

GCCGAAAAAGCTAACCCTGTCAACGATCTGTGTTACCCTGGTGACTTTAACGATTACGAA

GAACTGAAGCACCTGCTGTCTCGTATCAATCACTTCGAGAAAATCCAGATCATCCCGAAA

TCCTCCTGGAGCTCCCACGAAGCTTCTCTGGGCGTATCCTCCGCGTGCCCGTACCAGGGC

AAATCCTCTTTCTTTCGTAACGTTGTTTGGCTGATCAAGAAAAACTCCACCTACCCGACG

ATCAAGCGTAGCTATAATAACACCAACCAGGAAGACCTGCTGGTTCTGTGGGGCATCCAC

CATCCAAACGATGCTGCGGAACAGACCAAGCTGTACCAGAACCCGACCACCTACATCAGC

GTGGGCACCTCTACGCTGAACCAGCGTCTGGTACCGCGTATCGCAACCCGCAGCAAGGTA

AACGGTCAAAGCGGCCGCATGGAATTTTTCTGGACCATCCTGAAACCGAACGACGCAATC

AACTTCGAATCTAACGGCAATTTCATCGCTCCGGAGTATGCGTACAAAATCGTAAAGAAA

GGTGATAGCACTATCATGAAATCCGAGTAGTAA

SEQ ID NO：72 HA1-2mut VN

GGTGCGAAACCGCTGTCTCTGCGCGATTGCTCTGTGGCAGGCTGGCTGCTGGGCAACCCT

ATGTGTGACGAATTCATTAACGTTCCGGAATGGTCTGATATTGCCGAAAAAGCTAACCCT

GTCAACGATCTGTGTTACCCTGGTGACTTTAACGATTACGAAGAACTGAAGCACCTGCTG

TCTCGTATCAATCACTTCGAGAAAATCCAGATCATCCCGAAATCCTCCTGGAGCTCCCAC

GAAGCTTCTCTGGGCGTATCCTCCGCGTGCCCGTACCAGGGCAAATCCTCTTTCTTTCGT

AACGTTGTTTGGCTGATCAAGAAAAACTCCACCTACCCGACGATCAAGCGTAGCTATAAT

AACACCAACCAGGAAGACCTGCTGGTTCTGTGGGGCATCCACCATCCAAACGATGCTGCG

GAACAGACCAAGCTGTACCAGAACCCGACCACCTACATCAGCGTGGGCACCTCTACGCTG

AACCAGCGTCTGGTACCGCGTATCGCAACCCGCAGCAAGGTAAACGGTCAAAGCGGCCGC

ATGGAATTTTTCTGGACCATCCTGAAACCGAACGACGCAATCAACTTCGAATCTAACGGC

AATTTCATCGCTCCGGAGTATGCGTACAAAATCGTAAAGAAAGGTGATAGCACTATCATG

AAATCCGAGTAGTAA

SEQ ID NO：73 STF2.HA1-1 IND

ATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAA

TCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGC

GCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGT

CTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGC

GCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCT

AACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTG

AACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAG

GACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTG

AAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCAGAAAGCGTATGAT

GTGAAAGATACAGCAGTAACAACGAAAGCTTATGCCAATAATGGTACTACACTGGATGTA

TCGGGTCTTGATGATGCAGCTATTAAAGCGGCTACGGGTGGTACGAATGGTACGGCTTCT

GTAACCGGTGGTGCGGTTAAATTTGACGCAGATAATAACAAGTACTTTGTTACTATTGGT

GGCTTTACTGGTGCTGATGCCGCCAAAAATGGCGATTATGAAGTTAACGTTGCTACTGAC

GGTACAGTAACCCTTGCGGCTGGCGCAACTAAAACCACAATGCCTGCTGGTGCGACAACT

AAAACAGAAGTACAGGAGTTAAAAGATACACCGGCAGTTGTTTCAGCAGATGCTAAAAAT

GCCTTAATTGCTGGCGGCGTTGACGCTACCGATGCTAATGGCGCTGAGTTGGTCAAAATG

TCTTATACCGATAAAAATGGTAAGACAATTGAAGGCGGTTATGCGCTTAAAGCTGGCGAT

AAGTATTACGCCGCAGATTACGATGAAGCGACAGGAGCAATTAAAGCTAAAACCACAAGT

TATACTGCTGCTGACGGCACTACCAAAACAGCGGCTAACCAACTGGGTGGCGTAGACGGT

AAAACCGAAGTCGTTACTATCGACGGTAAAACCTACAATGCCAGCAAAGCCGCTGGTCAT

GATTTCAAAGCACAACCAGAGCTGGCGGAAGCAGCCGCTAAAACCACCGAAAACCCGCTG

CAGAAAATTGATGCCGCGCTGGCGCAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTA

CAAAACCGTTTCAACTCTGCTATCACCAACCTGGGCAATACCGTAAACAATCTGTCTGAA

GCGCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCG

CAGATTTTGCAGCAGGCCGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTCCCGCAGAAC

GTGCTGTCTCTGTTAGCGGAGAAAACGCATAACGGTAAACTGTGCGACCTGGACGGCGTG

AAGCCGCTGATCCTGCGTGACTGTTCTGTTGCTGGCTGGCTGCTGGGCAACCCGATGTGC

GATGAGTTCATCAACGTTCCGGAGTGGTCTTATATTGTTGAAAAAGCGAACCCGACCAAT

GACCTGTGCTACCCGGGCTCTTTTAATGACTACGAGGAGCTGAAACATCTGCTGTCTCGC

ATCAACCATTTCGAAAAAATTCAGATCATCCCGAAATCTTCCTGGAGCGATCACGAAGCT

TCTTCCGGCGTATCTTCTGCATGCCCTTACCTGGGTTCCCCGTCTTTCTTCCGTAACGTG

GTTTGGCTGATCAAGAAGAACTCTACGTACCCGACCATCAAGAAATCCTATAACAACACT

AACCAGGAAGATCTGCTGGTGCTGTGGGGTATCCATCATCCAAACGATGCAGCCGAACAG

ACCCGCCTGTACCAGAATCCGACCACGTACATCTCTATCGGCACTTCTACCCTGAATCAA

CGCCTGGTGCCGAAAATCGCAACTCGCAGCAAAGTCAACGGCCAATCTGGTCGCATGGAG

TTTTTCTGGACCATCCTGAAACCGAACGACGCGATTAACTTCGAAAGCAACGGCAACTTC

ATTGCACCGGAATATGCTTACAAGATCGTTAAAAAGGGTGATTCTGCGATCATGAAAAGC

GAGCTGGAATACGGCAACTGCAACACTAAATGCCAGACCCCGATGGGTGCAATCAATTCC

AGCATGCCATTTCACAACATCCACCCGCTGACCATCGGCGAGTGTCCAAAATACGTTAAA

TAATAG

SEQ ID NO:74 FOR primer

AGTGGCTGAGCCTGTTAGCGGAGAAAACGCATAACGGTAAACTGTG

SEQ ID NO：75 HA0s IND

GCTGAGCCTGTTACGTGATCAAATCTGCATCGGTTACCACGCAAACAACTCCACTGAACA

AGTGGATACGATCATGGAAAAGAACGTGACCGTGACCCACGCTCAAGATATCCTGGAGAA

AACGCATAACGGTAAACTGTGCGACCTGGACGGCGTGAAGCCGCTGATCCTGCGTGACTG

TTCTGTTGCTGGCTGGCTGCTGGGCAACCCGATGTGCGATGAGTTCATCAACGTTCCGGA

GTGGTCTTATATTGTTGAAAAAGCGAACCCGACCAATGACCTGTGCTACCCGGGCTCTTT

TAATGACTACGAGGAGCTGAAACATCTGCTGTCTCGCATCAACCATTTCGAAAAAATTCA

GATCATCCCGAAATCTTCCTGGAGCGATCACGAAGCTTCTTCCGGCGTATCTTCTGCATG

CCCTTACCTGGGTTCCCCGTCTTTCTTCCGTAACGTGGTTTGGCTGATCAAGAAGAACTC

TACGTACCCGACCATCAAGAAATCCTATAACAACACTAACCAGGAAGATCTGCTGGTGCT

GTGGGGTATCCATCATCCAAACGATGCAGCCGAACAGACCCGCCTGTACCAGAATCCGAC

CACGTACATCTCTATCGGCACTTCTACCCTGAATCAACGCCTGGTGCCGAAAATCGCAAC

TCGCAGCAAAGTCAACGGCCAATCTGGTCGCATGGAGTTTTTCTGGACCATCCTGAAACC

GAACGACGCGATTAACTTCGAAAGCAACGGCAACTTCATTGCACCGGAATATGCTTACAA

GATCGTTAAAAAGGGTGATTCTGCGATCATGAAAAGCGAGCTGGAATACGGCAACTGCAA

CACTAAATGCCAGACCCCGATGGGTGCAATCAATTCCAGCATGCCATTTCACAACATCCA

CCCGCTGACCATCGGCGAGTGTCCAAAATACGTTAAATCCAATCGTCTGGTGCTGGCAAC

GGGCCTGCGCAACTCTCCGCAACGTGAGTCTCGTCGCAAAAAGCGTGGCCTGTTTGGCGC

AATTGCGGGCTTCATCGAGGGCGGCTGGCAGGGTATGGTTGATGGTTGGTACGGTTATCA

CCATTCCAACGAGCAGGGCTCTGGTTACGCTGCGGACAAAGAGTCCACGCAGAAAGCGAT

CGATGGCGTCACCAACAAAGTGAACTCTATCATCGACAAGATGAACACTCAGTTCGAGGC

TGTGGGCCGTGAATTCAACAATCTGGAACGTCGCATCGAAAACCTGAACAAAAAGATGGA

AGACGGTTTCCTGGACGTATGGACCTATAACGCAGAGCTGCTGGTTCTGATGGAAAACGA

ACGTACTCTGGACTTCCACGATTCTAACGTTAAAAACCTGTACGACAAAGTTCGTCTGCA

GCTGCGCGATAATGCAAAAGAACTGGGTAACGGCTGCTTTGAATTTTACCACAAATGTGA

CAACGAATGCATGGAAAGCATCCGTAACGGTACCTACAATTACCCACAGTACTCCGAAGA

AGCGCGTCTGAAACGTGAAGAAATCTCCGGTGTAAAACTGGAATCCATTGGCACGTATCA

GATTCTGTCTATCTACTCCACGGTAGCGTCCTCTCTGGCGCTGGCAATTATGATGGCCGG

CCTGTCTCTGTGGATGTGCTCTAACGGCTCTCTGCAGTGCCGCATCTGCATCTAATAGGC

TGAGC

SEQ ID NO：76 STF2.HA1-2 IND

ATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAA

TCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGC

GCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGT

CTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGC

GCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCT

AACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTG

AACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAG

GACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTG

AAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCAGAAAGCGTATGAT

GTGAAAGATACAGCAGTAACAACGAAAGCTTATGCCAATAATGGTACTACACTGGATGTA

TCGGGTCTTGATGATGCAGCTATTAAAGCGGCTACGGGTGGTACGAATGGTACGGCTTCT

GTAACCGGTGGTGCGGTTAAATTTGACGCAGATAATAACAAGTACTTTGTTACTATTGGT

GGCTTTACTGGTGCTGATGCCGCCAAAAATGGCGATTATGAAGTTAACGTTGCTACTGAC

GGTACAGTAACCCTTGCGGCTGGCGCAACTAAAACCACAATGCCTGCTGGTGCGACAACT

AAAACAGAAGTACAGGAGTTAAAAGATACACCGGCAGTTGTTTCAGCAGATGCTAAAAAT

GCCTTAATTGCTGGCGGCGTTGACGCTACCGATGCTAATGGCGCTGAGTTGGTCAAAATG

TCTTATACCGATAAAAATGGTAAGACAATTGAAGGCGGTTATGCGCTTAAAGCTGGCGAT

AAGTATTACGCCGCAGATTACGATGAAGCGACAGGAGCAATTAAAGCTAAAACCACAAGT

TATACTGCTGCTGACGGCACTACCAAAACAGCGGCTAACCAACTGGGTGGCGTAGACGGT

AAAACCGAAGTCGTTACTATCGACGGTAAAACCTACAATGCCAGCAAAGCCGCTGGTCAT

GATTTCAAAGCACAACCAGAGCTGGCGGAAGCAGCCGCTAAAACCACCGAAAACCCGCTG

CAGAAAATTGATGCCGCGCTGGCGCAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTA

CAAAACCGTTTCAACTCTGCTATCACCAACCTGGGCAATACCGTAAACAATCTGTCTGAA

GCGCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCG

CAGATTTTGCAGCAGGCCGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTCCCGCAGAAC

GTGCTGAGCCTGTTAGCAGGTGTGAAACCGCTGATTCTGCGCGATTGTTCTGTTGCTGGC

TGGCTGCTGGGCAACCCGATGTGCGATGAGTTCATCAACGTTCCGGAGTGGTCTTATATT

GTTGAAAAAGCGAACCCGACCAATGACCTGTGCTACCCGGGCTCTTTTAATGACTACGAG

GAGCTGAAACATCTGCTGTCTCGCATCAACCATTTCGAAAAAATTCAGATCATCCCGAAA

TCTTCCTGGAGCGATCACGAAGCTTCTTCCGGCGTATCTTCTGCATGCCCTTACCTGGGT

TCCCCGTCTTTCTTCCGTAACGTGGTTTGGCTGATCAAGAAGAACTCTACGTACCCGACC

ATCAAGAAATCCTATAACAACACTAACCAGGAAGATCTGCTGGTGCTGTGGGGTATCCAT

CATCCAAACGATGCAGCCGAACAGACCCGCCTGTACCAGAATCCGACCACGTACATCTCT

ATCGGCACTTCTACCCTGAATCAACGCCTGGTGCCGAAAATCGCAACTCGCAGCAAAGTC

AACGGCCAATCTGGTCGCATGGAGTTTTTCTGGACCATCCTGAAACCGAACGACGCGATT

AACTTCGAAAGCAACGGCAACTTCATTGCACCGGAATATGCTTACAAGATCGTTAAAAAG

GGTGATTCTGCGATCATGAAAAGCGAGTAATAG

SEQ ID NO:77 REV primer

AGTCGCTCAGCCTATTACTCGCTTTTCATGATCGCAGAATCAC

SEQ ID NO：78 STF2.HA1-2mut IND

ATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAA

TCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGC

GCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGT

CTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGC

GCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCT

AACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTG

AACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAG

GACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTG

AAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCAGAAAGCGTATGAT

GTGAAAGATACAGCAGTAACAACGAAAGCTTATGCCAATAATGGTACTACACTGGATGTA

TCGGGTCTTGATGATGCAGCTATTAAAGCGGCTACGGGTGGTACGAATGGTACGGCTTCT

GTAACCGGTGGTGCGGTTAAATTTGACGCAGATAATAACAAGTACTTTGTTACTATTGGT

GGCTTTACTGGTGCTGATGCCGCCAAAAATGGCGATTATGAAGTTAACGTTGCTACTGAC

GGTACAGTAACCCTTGCGGCTGGCGCAACTAAAACCACAATGCCTGCTGGTGCGACAACT

AAAACAGAAGTACAGGAGTTAAAAGATACACCGGCAGTTGTTTCAGCAGATGCTAAAAAT

GCCTTAATTGCTGGCGGCGTTGACGCTACCGATGCTAATGGCGCTGAGTTGGTCAAAATG

TCTTATACCGATAAAAATGGTAAGACAATTGAAGGCGGTTATGCGCTTAAAGCTGGCGAT

AAGTATTACGCCGCAGATTACGATGAAGCGACAGGAGCAATTAAAGCTAAAACCACAAGT

TATACTGCTGCTGACGGCACTACCAAAACAGCGGCTAACCAACTGGGTGGCGTAGACGGT

AAAACCGAAGTCGTTACTATCGACGGTAAAACCTACAATGCCAGCAAAGCCGCTGGTCAT

GATTTCAAAGCACAACCAGAGCTGGCGGAAGCAGCCGCTAAAACCACCGAAAACCCGCTG

CAGAAAATTGATGCCGCGCTGGCGCAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTA

CAAAACCGTTTCAACTCTGCTATCACCAACCTGGGCAATACCGTAAACAATCTGTCTGAA

GCGCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCG

CAGATTTTGCAGCAGGCCGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTCCCGCAGAAC

GTGCTGTCTCTGTTAGCGGGCGCAAAGCCGCTGTCTCTGCGTGACTGTTCTGTTGCTGGC

TGGCTGCTGGGCAACCCGATGTGCGATGAGTTCATCAACGTTCCGGAGTGGTCTGATATT

GCGGAAAAAGCGAACCCGACCAATGACCTGTGCTACCCGGGCTCTTTTAATGACTACGAG

GAGCTGAAACATCTGCTGTCTCGCATCAACCATTTCGAAAAAATTCAGATCATCCCGAAA

TCTTCCTGGAGCGATCACGAAGCTTCTTCCGGCGTATCTTCTGCATGCCCTTACCTGGGT

TCCCCGTCTTTCTTCCGTAACGTGGTTTGGCTGATCAAGAAGAACTCTACGTACCCGACC

ATCAAGAAATCCTATAACAACACTAACCAGGAAGATCTGCTGGTGCTGTGGGGTATCCAT

CATCCAAACGATGCAGCCGAACAGACCCGCCTGTACCAGAATCCGACCACGTACATCTCT

ATCGGCACTTCTACCCTGAATCAACGCCTGGTGCCGAAAATCGCAACTCGCAGCAAAGTC

AACGGCCAATCTGGTCGCATGGAGTTTTTCTGGACCATCCTGAAACCGAACGACGCGATT

AACTTCGAAAGCAACGGCAACTTCATTGCACCGGAATATGCTTACAAGATCGTTAAAAAG

GGTGATTCTGCGATCATGAAAAGCGAGTAATAG

SEQ ID 79 HA1-2mut IND

GGCGCAAAGCCGCTGTCTCTGCGTGACTGTTCTGTTGCTGGCTGGCTGCTGGGCAACCCG

ATGTGCGATGAGTTCATCAACGTTCCGGAGTGGTCTGATATTGCGGAAAAAGCGAACCCG

ACCAATGACCTGTGCTACCCGGGCTCTTTTAATGACTACGAGGAGCTGAAACATCTGCTG

TCTCGCATCAACCATTTCGAAAAAATTCAGATCATCCCGAAATCTTCCTGGAGCGATCAC

GAAGCTTCTTCCGGCGTATCTTCTGCATGCCCTTACCTGGGTTCCCCGTCTTTCTTCCGT

AACGTGGTTTGGCTGATCAAGAAGAACTCTACGTACCCGACCATCAAGAAATCCTATAAC

AACACTAACCAGGAAGATCTGCTGGTGCTGTGGGGTATCCATCATCCAAACGATGCAGCC

GAACAGACCCGCCTGTACCAGAATCCGACCACGTACATCTCTATCGGCACTTCTACCCTG

AATCAACGCCTGGTGCCGAAAATCGCAACTCGCAGCAAAGTCAACGGCCAATCTGGTCGC

ATGGAGTTTTTCTGGACCATCCTGAAACCGAACGACGCGATTAACTTCGAAAGCAACGGC

AACTTCATTGCACCGGAATATGCTTACAAGATCGTTAAAAAGGGTGATTCTGCGATCATG

AAAAGCGAGTAATAGGCTGAGC

SEQ ID NO：80 STF2.HA1-1 NC

ATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAA

TCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGC

GCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGT

CTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGC

GCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCT

AACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTG

AACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAG

GACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTG

AAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCAGAAAGCGTATGAT

GTGAAAGATACAGCAGTAACAACGAAAGCTTATGCCAATAATGGTACTACACTGGATGTA

TCGGGTCTTGATGATGCAGCTATTAAAGCGGCTACGGGTGGTACGAATGGTACGGCTTCT

GTAACCGGTGGTGCGGTTAAATTTGACGCAGATAATAACAAGTACTTTGTTACTATTGGT

GGCTTTACTGGTGCTGATGCCGCCAAAAATGGCGATTATGAAGTTAACGTTGCTACTGAC

GGTACAGTAACCCTTGCGGCTGGCGCAACTAAAACCACAATGCCTGCTGGTGCGACAACT

AAAACAGAAGTACAGGAGTTAAAAGATACACCGGCAGTTGTTTCAGCAGATGCTAAAAAT

GCCTTAATTGCTGGCGGCGTTGACGCTACCGATGCTAATGGCGCTGAGTTGGTCAAAATG

TCTTATACCGATAAAAATGGTAAGACAATTGAAGGCGGTTATGCGCTTAAAGCTGGCGAT

AAGTATTACGCCGCAGATTACGATGAAGCGACAGGAGCAATTAAAGCTAAAACCACAAGT

TATACTGCTGCTGACGGCACTACCAAAACAGCGGCTAACCAACTGGGTGGCGTAGACGGT

AAAACCGAAGTCGTTACTATCGACGGTAAAACCTACAATGCCAGCAAAGCCGCTGGTCAT

GATTTCAAAGCACAACCAGAGCTGGCGGAAGCAGCCGCTAAAACCACCGAAAACCCGCTG

CAGAAAATTGATGCCGCGCTGGCGCAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTA

CAAAACCGTTTCAACTCTGCTATCACCAACCTGGGCAATACCGTAAACAATCTGTCTGAA

GCGCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCG

CAGATTTTGCAGCAGGCCGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTCCCGCAGAAC

GTGCTGAGCCTGTTAGCGTCCCACAACGGTAAACTGTGTCTGCTGAAAGGCATCGCACCG

CTGCAGCTGGGTAACTGTAGCGTTGCAGGTTGGATCCTGGGTAACCCGGAGTGCGAACTG

CTGATTTCCAAGGAGAGCTGGTCTTACATTGTCGAAACGCCGAATCCGGAAAACGGCACT

TGTTATCCGGGTTACTTCGCCGATTACGAAGAACTGCGTGAACAACTGTCTTCCGTGAGC

TCTTTCGAACGTTTCGAGATCTTCCCGAAAGAGAGCAGCTGGCCAAACCACACTGTTACC

GGTGTGTCTGCGAGCTGTTCTCACAACGGCAAGTCTTCTTTCTACCGCAACCTGCTGTGG

CTGACGGGTAAGAATGGCCTGTATCCGAACCTGTCTAAATCTTACGTAAACAACAAAGAG

AAAGAGGTGCTGGTCCTGTGGGGCGTACACCATCCACCAAATATCGGCAACCAGCGCGCC

CTGTACCACACCGAAAACGCTTATGTGTCCGTGGTGAGCTCCCATTACAGCCGTCGTTTT

ACTCCGGAGATTGCCAAACGTCCGAAAGTTCGTGATCAGGAAGGCCGTATTAACTACTAC

TGGACTCTGCTGGAGCCGGGCGATACCATCATTTTCGAGGCAAACGGCAACCTGATTGCG

CCATGGTACGCGTTCGCCCTGAGCCGTGGTTTTGGCTCCGGTATTATCACCTCTAACGCG

CCAATGGACGAATGCGACGCGAAATGCCAAACGCCGCAGGGCGCAATCAACAGCAGCCTG

CCGTTCCAGAACGTTCACCCGGTTACCATCGGCGAATGCCCTAAATACGTGCGCTAATAG

SEQ ID NO:81 FOR primer

AACCAGGTCCCGCAGAACGTGCTGAGCCTGTTAGCGTCCCACAACGGTAAACTGTGTCTG

CTGAAAGGCATCGCACCGCTGCAG

SEQ ID NO:82 REV primer

GAACAGCTCAGTTCCTACGAGCTCAGCCTATTAGCGCACGTATTTAGGGCATTCGCCGATG

GTAACCGGGTGAACGTT

SEQ ID NO：83 HA0s NC

GACACGATCTGTATTGGTTATCATGCAAACAACTCTACTGACACTGTAGATACTGTGCTG

GAAAAGAACGTAACCGTTACCCACAGCGTTAACCTGCTGGAAGATTCCCACAACGGTAAA

CTGTGTCTGCTGAAAGGCATCGCACCGCTGCAGCTGGGTAACTGTAGCGTTGCAGGTTGG

ATCCTGGGTAACCCGGAGTGCGAACTGCTGATTTCCAAGGAGAGCTGGTCTTACATTGTC

GAAACGCCGAATCCGGAAAACGGCACTTGTTATCCGGGTTACTTCGCCGATTACGAAGAA

CTGCGTGAACAACTGTCTTCCGTGAGCTCTTTCGAACGTTTCGAGATCTTCCCGAAAGAG

AGCAGCTGGCCAAACCACACTGTTACCGGTGTGTCTGCGAGCTGTTCTCACAACGGCAAG

TCTTCTTTCTACCGCAACCTGCTGTGGCTGACGGGTAAGAATGGCCTGTATCCGAACCTG

TCTAAATCTTACGTAAACAACAAAGAGAAAGAGGTGCTGGTCCTGTGGGGCGTACACCAT

CCACCAAATATCGGCAACCAGCGCGCCCTGTACCACACCGAAAACGCTTATGTGTCCGTG

GTGAGCTCCCATTACAGCCGTCGTTTTACTCCGGAGATTGCCAAACGTCCGAAAGTTCGT

GATCAGGAAGGCCGTATTAACTACTACTGGACTCTGCTGGAGCCGGGCGATACCATCATT

TTCGAGGCAAACGGCAACCTGATTGCGCCATGGTACGCGTTCGCCCTGAGCCGTGGTTTT

GGCTCCGGTATTATCACCTCTAACGCGCCAATGGACGAATGCGACGCGAAATGCCAAACG

CCGCAGGGCGCAATCAACAGCAGCCTGCCGTTCCAGAACGTTCACCCGGTTACCATCGGC

GAATGCCCTAAATACGTGCGCTCCGCCAAACTGCGCATGGTTACTGGTCTGCGTAACATC

CCGAGCATTCAGTCTCGCGGTCTGTTCGGTGCGATCGCGGGCTTCATTGAAGGCGGTTGG

ACCGGTATGGTGGATGGTTGGTACGGCTACCATCACCAGAACGAACAGGGTAGCGGTTAC

GCTGCCGACCAGAAATCCACCCAGAACGCTATTAACGGTATCACCAACAAAGTTAACAGC

GTAATTGAGAAGATGAACACGCAGTTCACCGCCGTAGGTAAGGAATTCAACAAGCTGGAA

CGTCGCATGGAAAACCTGAACAAAAAGGTGGACGACGGCTTTCTGGACATCTGGACCTAC

AACGCTGAACTGCTGGTGCTGCTGGAAAACGAACGTACCCTGGATTTCCACGACTCTAAT

GTTAAAAACCTGTACGAAAAGGTCAAGTCTCAACTGAAAAACAATGCGAAGGAAATCGGC

AACGGCTGTTTCGAATTCTACCATAAATGCAACAACGAATGCATGGAATCCGTTAAAAAC

GGTACCTATGACTACCCTAAATACTCCGAAGAAAGCAAACTGAACCGCGAGAAAATCGAT

GGTGTAAAACTGGAATCTATGGGTGTTTACCAGATCCTGGCGATCTACTCCACGGTAGCC

AGCAGCCTGGTTCTGCTGGTTAGCCTGGGTGCAATTAGCTTCTGGATGTGCTCTAACGGC

AGCCTGCAATGCCGCATCTGTAATAGGCTGAGC

SEQ ID NO：84 STF2HA1-2 NC

ATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAA

TCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGC

GCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGT

CTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGC

GCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCT

AACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTG

AACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAG

GACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTG

AAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCAGAAAGCGTATGAT

GTGAAAGATACAGCAGTAACAACGAAAGCTTATGCCAATAATGGTACTACACTGGATGTA

TCGGGTCTTGATGATGCAGCTATTAAAGCGGCTACGGGTGGTACGAATGGTACGGCTTCT

GTAACCGGTGGTGCGGTTAAATTTGACGCAGATATATAACAAGTACTTTGTTACTATTGGT

GGCTTTACTGGTGCTGATGCCGCCAAAAATGGCGATTATGAAGTTAACGTTGCTACTGAC

GGTACAGTAACCCTTGCGGCTGGCGCAACTAAAACCACAATGCCTGCTGGTGCGACAACT

AAAACAGAAGTACAGGAGTTAAAAGATACACCGGCAGTTGTTTCAGCAGATGCTAAAAAT

GCCTTAATTGCTGGCGGCGTTGACGCTACCGATGCTAATGGCGCTGAGTTGGTCAAAATG

TCTTATACCGATAAAAATGGTAAGACAATTGAAGGCGGTTATGCGCTTAAAGCTGGCGAT

AAGTATTACGCCGCAGATTACGATGAAGCGACAGGAGCAATTAAAGCTAAAACCACAAGT

TATACTGCTGCTGACGGCACTACCAAAACAGCGGCTAACCAACTGGGTGGCGTAGACGGT

AAAACCGAAGTCGTTACTATCGACGGTAAAACCTACAATGCCAGCAAAGCCGCTGGTCAT

GATTTCAAAGCACAACCAGAGCTGGCGGAAGCAGCCGCTAAAACCACCGAAAACCCGCTG

CAGAAAATTGATGCCGCGCTGGCGCAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTA

CAAAACCGTTTCAACTCTGCTATCACCAACCTGGGCAATACCGTAAACAATCTGTCTGAA

GCGCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCG

CAGATTTTGCAGCAGGCCGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTCCCGCAGAAC

GTGCTGAGCCTGTTAGCGAAAGGCATCGCACCGCTGCAGCTGGGTAACTGTAGCGTTGCA

GGTTGGATCCTGGGTAACCCGGAGTGCGAACTGCTGATTTCCAAGGAGAGCTGGTCTTAC

ATTGTCGAAACGCCGAATCCGGAAAACGGCACTTGTTATCCGGGTTACTTCGCCGATTAC

GAAGAACTGCGTGAACAACTGTCTTCCGTGAGCTCTTTCGAACGTTTCGAGATCTTCCCG

AAAGAGAGCAGCTGGCCAAACCACACTGTTACCGGTGTGTCTGCGAGCTGTTCTCACAAC

GGCAAGTCTTCTTTCTACCGCAACCTGCTGTGGCTGACGGGTAAGAATGGCCTGTATCCG

AACCTGTCTAAATCTTACGTAAACAACAAAGAGAAAGAGGTGCTGGTCCTGTGGGGCGTA

CACCATCCACCAAATATCGGCAACCAGCGCGCCCTGTACCACACCGAAAACGCTTATGTG

TCCGTGGTGAGCTCCCATTACAGCCGTCGTTTTACTCCGGAGATTGCCAAACGTCCGAAA

GTTCGTGATCAGGAAGGCCGTATTAACTACTACTGGACTCTGCTGGAGCCGGGCGATACC

ATCATTTTCGAGGCAAACGGCAACCTGATTGCGCCATGGTACGCGTTCGCCCTGAGCCGT

GGTTTTGGCTCCGGTATTATCACCTCTTAATAA

SEQ ID NO:85 FOR primer

AACCAGGTCCCGCAGAACGTGCTGAGCCTGTTAGCGAAAGGCATCGCACCGCTGCAGCTG

GGTAACTGTAGCGTTGCAGGTTGG

SEQ ID NO:86 REV primer

GAACAGCTCAGTTCCTACGAGCTCAGCCTATTAAGAGGTGATAATACCGGAGCCAAAACC

ACGGCTCAGGGCGAACGCGTACCA

SEQ ID NO：87 STF2.HA1-2mut NC

ATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAA

TCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGC

GCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGT

CTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGC

GCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCT

AACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTG

AACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAG

GACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTG

AAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCAGAAAGCGTATGAT

GTGAAAGATACAGCAGTAACAACGAAAGCTTATGCCAATAATGGTACTACACTGGATGTA

TCGGGTCTTGATGATGCAGCTATTAAAGCGGCTACGGGTGGTACGAATGGTACGGCTTCT

GTAACCGGTGGTGCGGTTAAATTTGACGCAGATAATAACAAGTACTTTGTTACTATTGGT

GGCTTTACTGGTGCTGATGCCGCCAAAAATGGCGATTATGAAGTTAACGTTGCTACTGAC

GGTACAGTAACCCTTGCGGCTGGCGCAACTAAAACCACAATGCCTGCTGGTGCGACAACT

AAAACAGAAGTACAGGAGTTAAAAGATACACCGGCAGTTGTTTCAGCAGATGCTAAAAAT

GCCTTAATTGCTGGCGGCGTTGACGCTACCGATGCTAATGGCGCTGAGTTGGTCAAAATG

TCTTATACCGATAAAAATGGTAAGACAATTGAAGGCGGTTATGCGCTTAAAGCTGGCGAT

AAGTATTACGCCGCAGATTACGATGAAGCGACAGGAGCAATTAAAGCTAAAACCACAAGT

TATACTGCTGCTGACGGCACTACCAAAACAGCGGCTAACCAACTGGGTGGCGTAGACGGT

AAAACCGAAGTCGTTACTATCGACGGTAAAACCTACAATGCCAGCAAAGCCGCTGGTCAT

GATTTCAAAGCACAACCAGAGCTGGCGGAAGCAGCCGCTAAAACCACCGAAAACCCGCTG

CAGAAAATTGATGCCGCGCTGGCGCAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTA

CAAAACCGTTTCAACTCTGCTATCACCAACCTGGGCAATACCGTAAACAATCTGTCTGAA

GCGCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCG

CAGATTTTGCAGCAGGCCGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTCCCGCAGAAC

GTGCTGAGCCTGTTAGCGAAAGGCGCGGCACCGCTGCAGCTGGGTAACTGTAGCGTTGCA

GGTTGGATCCTGGGTAACCCGGAGTGCGAACTGCTGATTTCCAAGGAGAGCTGGTCTGAT

ATTGCAGAAACGCCGAATCCGGAAAACGGCACTTGTTATCCGGGTTACTTCGCCGATTAC

GAAGAACTGCGTGAACAACTGTCTTCCGTGAGCTCTTTCGAACGTTTCGAGATCTTCCCG

AAAGAGAGCAGCTGGCCAAACCACACTGTTACCGGTGTGTCTGCGAGCTGTTCTCACAAC

GGCAAGTCTTCTTTCTACCGCAACCTGCTGTGGCTGACGGGTAAGAATGGCCTGTATCCG

AACCTGTCTAAATCTTACGTAAACAACAAAGAGAAAGAGGTGCTGGTCCTGTGGGGCGTA

CACCATCCACCAAATATCGGCAACCAGCGCGCCCTGTACCACACCGAAAACGCTTATGTG

TCCGTGGTGAGCTCCCATTACAGCCGTCGTTTTACTCCGGAGATTGCCAAACGTCCGAAA

GTTCGTGATCAGGAAGGCCGTATTAACTACTACTGGACTCTGCTGGAGCCGGGCGATACC

ATCATTTTCGAGGCAAACGGCAACCTGATTGCGCCATGGTACGCGTTCGCCCTGAGCCGT

GGTTTTGGCTCCGGTATTATCACCTCTTAATAG

SEQ ID NO：88 HA1-2mut NC

GCTGAGCCTGTTAGCGAAAGGCGCGGCACCGCTGCAGCTGGGTAACTGTAGCGTTGCAGG

TTGGATCCTGGGTAACCCGGAGTGCGAACTGCTGATTTCCAAGGAGAGCTGGTCTGATAT

TGCAGAAACGCCGAATCCGGAAAACGGCACTTGTTATCCGGGTTACTTCGCCGATTACGA

AGAACTGCGTGAACAACTGTCTTCCGTGAGCTCTTTCGAACGTTTCGAGATCTTCCCGAA

AGAGAGCAGCTGGCCAAACCACACTGTTACCGGTGTGTCTGCGAGCTGTTCTCACAACGG

CAAGTCTTCTTTCTACCGCAACCTGCTGTGGCTGACGGGTAAGAATGGCCTGTATCCGAA

CCTGTCTAAATCTTACGTAAACAACAAAGAGAAAGAGGTGCTGGTCCTGTGGGGCGTACA

CCATCCACCAAATATCGGCAACCAGCGCGCCCTGTACCACACCGAAAACGCTTATGTGTC

CGTGGTGAGCTCCCATTACAGCCGTCGTTTTACTCCGGAGATTGCCAAACGTCCGAAAGT

TCGTGATCAGGAAGGCCGTATTAACTACTACTGGACTCTGCTGGAGCCGGGCGATACCAT

CATTTTCGAGGCAAACGGCAACCTGATTGCGCCATGGTACGCGTTCGCCCTGAGCCGTGG

TTTTGGCTCCGGTATTATCACCTCTTAATAGGCTGAGC

SEQ ID NO：89 STF2.HA1-1his(PR8)

MAQVTNTNSLSLLTQNNLNKSQSALGTAIERLSSGLRTNSAKDDAAGQAIANRFTANIKGLTQASR

NANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLNEIDRVSGQTQFN

GVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDSLNVQKAYDVKDTAVTTKAYANNGTTL

DVSGLDDAAIKAATGGTNGTASVTGGAVKFDADNNKYFVTIGGFTGADAAKNGDYEVNVATDG

TVTLAAGATKTTMPAGATTKTEVQELKDTPAVVSADAKNALIAGGVDATDANGAELVKMSYTDK

NGKTIEGGYALKAGDKYYAADYDEATGAIKAKTTSYTAADGTTKTAANQLGGVDGKTEVVTIDG

KTYNASKAAGHDFKAQPELAEAAAKTTENPLQKIDAALAQVDALRSDLGAVQNRFNSAITNLGN

TVNNLSEARSRIEDSDYATEVSNMSRAQILQQAGTSVLAQANQVPQNVLSLLASHNGKLCRLKGI

APLQLGKCNIAGWLLGNPECDPLLPVRSWSYIVETPNSENGICYPGDFIDYEELREQLSSVSSFERF

EIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTEKEGSYPKLKNSYVNKKGKEVLVLWGIH

HPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQAGRMNYYWTLLKPGDTIIFEAN

GNLIAPMYAFALSRGFGSGIITSNASMHECNTKCQTPLGAINSSLPYQNIHPVTIGECPKYVRHHHH

HH

SEQ ID NO：90 STF2.HA1-2(PR8)

MAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANRFTANIKGLTQASR

NANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLNEIDRVSGQTQFN

GVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDSLNVQKAYDVKDTAVTTKAYANNGTTL

DVSGLDDAAIKAATGGTNGTASVTGGAVKFDADNNKYFVTIGGFTGADAAKNGDYEVNVATDG

TVTLAAGATKTTMPAGATTKTEVQELKDTPAVVSADAKNALIAGGVDATDANGAELVKMSYTDK

NGKTIEGGYALKAGDKYYAADYDEATGAIKAKTTSYTAADGTTKTAANQLGGVDGKTEVVTIDG

KTYNASKAAGHDFKAQPELAEAAAKTTENPLQKIDAALAQVDALRSDLGAVQNRFNSAITNLGN

TVNNLSEARSRIEDSDYATEVSNMSRAQILQQAGTSVLAQANQVPQNVLSLLAKGIAPLQLGKCNI

AGWLLGNPECDPLLPVRSWSYIVETPNSENGICYPGDFIDYEELREQLSSVSSFERFEIFPKESSWP

NHNTNGVTAACSHEGKSSFYRNLLWLTEKEGSYPKLKNSYVNKKGKEVLVLWGIHHPPNSKEQQ

NLYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQAGRMNYYWTLLKPGDTIIFEANGNLIAPMYA

FALSRGFGSGIITS

SEQ ID NO：91 STF2.HA1-2mut(PR8)

MAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANRFTANIKGLTQASR

NANDGISIAQTTEGALNEINNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLNEIDRVSGQTQFN

GVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDSLNVQKAYDVKDTAVTTKAYANNGTTL

DVSGLDDAAIKAATGGTNGTASVTGGAVKFDADNNKYFVTIGGFTGADAAKNGDYEVNVATDG

TVTLAAGATKTTMPAGATTKTEVQELKDTPAVVSADAKNALIAGGVDATDANGAELVKMSYTDK

NGKTIEGGYALKAGDKYYAADYDEATGAIKAKTTSYTAADGTTKTAANQLGGVDGKTEVVTIDG

KTYNASKAAGHDFKAQPELAEAAAKTTENPLQKIDAALAQVDALRSDLGAVQNRFNSAITNLGN

TVNNLSEARSRIEDSDYATEVSNMSRAQILQQAGTSVLAQANQVPQNVLSLLAKGAAPLQLGKC

NIAGWLLGNPECDPLLPVRSWSDIAETPNSENGICYPGDFIDYEELREQLSSVSSFERFEIFPKESSW

PNHNTNGVTAACSHEGKSSFYRNLLWLTEKEGSYPKLKNSYVNKKGKEVLVLWGIHHPPNSKEQ

QNLYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQAGRMNYYWTLLKPGDTIIFEANGNLIAPMY

AFALSRGFGSGIITS

SEQ ID NO：92 STF2.HA1-3(PR8)

MAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANRFTANIKGLTQASR

NANDGISIAQTTEGALNEINNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLNEIDRVSGQTQFN

GVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDSLNVQKAYDVKDTAVTTKAYANNGTTL

DVSGLDDAAIKAATGGTNGTASVTGGAVKFDADNNKYFVTIGGFTGADAAKNGDYEVNVATDG

TVTLAAGATKTTMPAGATTKTEVQELKDTPAVVSADAKNALIAGGVDATDANGAELVKMSYTDK

NGKTIEGGYALKAGDKYYAADYDEATGAIKAKTTSYTAADGTTKTAANQLGGVDGKTEVVTIDG

KTYNASKAAGHDFKAQPELAEAAAKTTENPLQKIDAALAQVDALRSDLGAVQNRFNSAITNLGN

TVNNLSEARSRIEDSDYATEVSNMSRAQILQQAGTSVLAQANQVPQNVLSLLANSENGICYPGDFI

DYEELREQLSSVSSFERFEIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTEKEGSYPKLKN

SYVNKKGKEVLVLWGIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQAGRM

NYYWTLLKPGDTIIFEANGNLIAPMYAFALSRG

SEQ ID NO：93 HA1-2his(PR8)

KGIAPLQLGKCNIAGWLLGNPECDPLLPVRSWSYIVETPNSENGICYPGDFIDYEELREQLSSVSSF

ERFEIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTEKEGSYPKLKNSYVNKKGKEVLVLW

GIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQAGRMNYYWTLLKPGDTIIF

EANGNLIAPMYAFALSRGFGSGIITSHHHHHH

SEQ ID NO：94 STF2.4xM2e

MAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANRFTANIKGLTQASR

NANDGISIAQTTEGALNEINNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLNEIDRVSGQTQFN

GVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDSLNVQKAYDVKDTAVTTKAYANNGTTL

DVSGLDDAAIKAATGGTNGTASVTGGAVKFDADNNKYFVTIGGFTGADAAKNGDYEVNVATDG

TVTLAAGATKTTMPAGATTKTEVQELKDTPAVVSADAKNALIAGGVDATDANGAELVKMSYTDK

NGKTIEGGYALKAGDKYYAADYDEATGAIKAKTTSYTAADGTTKTAANQLGGVDGKTEVVTIDG

KTYNASKAAGHDFKAQPELAEAAAKTTENPLQKIDAALAQVDALRSDLGAVQNRFNSAITNLGN

TVNNLSEARSRIEDSDYATEVSNMSRAQILQQAGTSVLAQANQVPQNVLSLLRLSLLTEVETPIRN

EWGSRSNDSSDPLESLLTEVETPIRNEWGSRSNDSSDPGSSLLTEVETPIRNEWGSRSNDSSDPELS

LLTEVETPIRNEWGSRSNDSSDPSR

SEQ ID NO：95 STF2.HA1-2(VN)

MAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANRFTANIKGLTQASR

NANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLNEIDRVSGQTQFN

GVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDSLNVQKAYDVKDTAVTTKAYANNGTTL

DVSGLDDAAIKAATGGTNGTASVTGGAVKFDADNNKYFVTIGGFTGADAAKNGDYEVNVATDG

TVTLAAGATKTTMPAGATTKTEVQELKDTPAVVSADAKNALIAGGVDATDANGAELVKMSYTDK

NGKTIEGGYALKAGDKYYAADYDEATGAIKAKTTSYTAADGTTKTAANQLGGVDGKTEVVTIDG

KTYNASKAAGHDFKAQPELAEAAAKTTENPLQKIDAALAQVDALRSDLGAVQNRFNSAITNLGN

TVNNLSEARSRIEDSDYATEVSNMSRAQILQQAGTSVLAQANQVPQNVLSLLAGVKPLILRDCSV

AGWLLGNPMCDEFINVPEWSYIVEKANPVNDLCYPGDFNDYEELKHLLSRINHFEKIQIIPKSSWS

SHEASLGVSSACPYQGKSSFFRNVVMLIKKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQT

KLYQNPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYK

IVKKGDSTIMKSE

SEQ ID NO：96 STF2

MAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANRFTANIKGLTQASR

NANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLNEIDRVSGQTQFN

GVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDSLNVQKAYDVKDTAVTTKAYANNGTTL

DVSGLDDAAIKAATGGTNGTASVTGGAVKFDADNNKYFVTIGGFTGADAAKNGDYEVNVATDG

TVTLAAGATKTTMPAGATTKTEVQELKDTPAVVSADAKNALIAGGVDATDANGAELVKMSYTDK

NGKTIEGGYALKAGDKYYAADYDEATGAIKAKTTSYTAADGTTKTAANQLGGVDGKTEVVTIDG

KTYNASKAAGHDFKAQPELAEAAAKTTENPLQKIDAALAQVDALRSDLGAVQNRFNSAITNLGN

TVNNLSEARSRIEDSDYATEVSNMSRAQILQQAGTSVLAQANQVPQNVLSLLA

SEQ ID NO：97 STF2.Blp.wt

ATGAAATTCTTAGTCAACGTTGCCCTTGTTTTTATGGTCGTATACATTTCTTACATCTAT

GCGGCCCAGGTTATCAATACCAACTCCCTGTCGTTGCTCACCCAAAATAACCTTAATAAA

AGCCAGAGCGCACTGGGAACCGCCATAGAACGCCTCTCAAGCGGCCTCCGGATCAATTCT

GCAAAAGACGACGCCGCCGGTCAGGCCATCGCAAACCGCTTTACCGCCAATATCAAGGGA

CTGACGCAGGCTTCGAGGAATGCTAACGATGGAATAAGCATCGCTCAAACCACGGAGGGC

GCCCTGAACGAGATCAACAACAACCTACAGCGCGTCAGGGAGCTCGCAGTGCAGTCCGCC

AATTCGACCAACTCGCAGTCGGACCTGGACTCGATCCAAGCCGAAATCACCCAGCGCCTG

AATGAGATTGACCGGGTGAGCGGTCAGACACAGTTTAACGGCGTGAAGGTACTTGCACAG

GATAACACACTTACGATACAGGTGGGCGCCAACGATGGTGAAACCATAGACATTGATCTC

AAACAGATTAACAGCCAGACGCTCGGGTTGGATAGCCTGAATGTGCAAAAGGCGTACGAC

GTGAAAGACACGGCGGTCACTACCAAAGCCTACGCTAACAATGGCACTACCTTGGATGTG

AGCGGATTGGATGATGCAGCAATCAAGGCTGCTACCGGCGGTACGAACGGAACCGCGTCC

GTGACCGGCGGTGCCGTGAAGTTCGATGCTGACAACAATAAGTATTTCGTCACCATTGGA

GGCTTTACTGGCGCCGACGCAGCAAAGAACGGCGACTATGAAGTGAACGTGGCAACCGAT

GGAACCGTGACGCTGGCCGCTGGTGCCACCAAGACCACCATGCCAGCCGGCGCCACAACT

AAGACCGAGGTGCAGGAGTTAAAGGACACCCCCGCGGTGGTTAGCGCAGATGCCAAAAAC

GCGTTGATCGCCGGCGGAGTGGATGCAACTGATGCTAATGGTGCGGAGCTGGTTAAAATG

TCGTATACAGACAAGAATGGTAAGACGATCGAGGGCGGTTATGCCCTTAAGGCAGGAGAT

AAGTATTACGCTGCTGATTACGATGAGGCGACGGGAGCTATTAAGGCCAAGACAACGTCA

TACACGGCGGCGGACGGAACGACTAAGACGGCTGCCAATCAGTTGGGAGGGGTTGACGGG

AAGACAGAGGTCGTTACGATCGATGGCAAGACATACAACGCCTCCAAGGCCGCTGGCCAC

GATTTCAAAGCTCAACCCGAACTGGCCGAGGCCGCGGCGAAAACAACTGAGAACCCGTTG

CAGAAGATTGATGCGGCCCTGGCGCAAGTAGATGCCCTGCGCTCAGACCTGGGCGCCGTT

CAAAATCGATTCAATTCCGCGATTACAAACCTGGGCAATACAGTAAACAATCTATCCGAG

GCCAGATCCCGCATTGAAGACTCCGACTACGCGACAGAAGTAAGTAACATGAGTCGTGCC

CAGATTCTGCAGCAGGCCGGCACTAGTGTCCTGGCCCAGGCCAATCAAGTCCCGCAGAAT

GTGCTGAGCCTACTACGAGAGTTCAGTCGTTACCCAGCCCAATGGCGGCCGCTC

SEQ ID NO：98 STF2.Blp.ng

ATGAAATTCTTAGTCAACGTTGCCCTTGTTTTTATGGTCGTATACATTTCTTACATCTAT

GCGGCCCAAGTGATCAACACCAACTCCCTGTCCCTGCTCACTCAAAACAACCTCCAGAAA

TCTCAGTCTGCTCTCGGTACTGCTATCGAGCGTCTCTCCTCCGGCCTGAGGATCAACTCC

GCTAAAGATGACGCCGCCGGACAAGCTATCGCTAACCGCTTTACTGCCAACATTAAAGGC

CTGACCCAAGCCTCTAGGAACGCCAATGATGCGCATTTCTATCGCTCAAACCACCGAAGGC

GCCCTGAACGAAATCAATAATAACCTGCAACGTGTCCGCGAACTCGCCGTCCAGTCCGCC

CAATCTACCAACTCTCAGTCTGACCTGGACTCTATCCAAGCTGAGATCACCCAAAGGCTC

AACGAGATCGATCGTGTGTCTGGTCAAACTCAGTTTAACGGCGTCAAAGTGCTGGCTCAA

GACAACACACTCACCATCCAAGTAGGAGCCAATGACGGCGAGACAATCGATATCGACCTG

AAGCAGATCAATTCTCAAACTCTGGGCCTGGACTCCCTGAACGTCCAAAAGGCTTACGAC

GTGAAGGACACCGCTGTGACAACCAAAGCCTATGCTAATCAAGGAACAACCCTGGACGTG

TCCGGACTCGATGACGCCGCTATCAAGGCCGCTACTGGCGGCACTCAGGGAACTGCCTCT

GTGACCGGAGGCGCTGTGAAGTTCGACGCCGATAACAACAAATACTTCGTCACTATTGGT

GGTTTCACTGGCGCTGACGCCGCTAAGAACGGTGACTACGAGGTCAACGTCGCCACTGAC

GGAACAGTGACACTGGCCGCTGGCGCTACCAAGACCACAATGCCTGCTGGTGCTACTACT

AAGACAGAGGTGCAGGAACTCAAGGACACCCCTGCCGTGGTGTCCGCCGATGCTAAGAAC

GCTCTCATTGCTGGTGGTGTCGACGCTACGGACGCCAACGGAGCCGAGCTTGTGAAAATG

TCCTACACCGACAAGAACGGAAAGACTATCGAGGGAGGTTACGCTCTGAAGGCTGGCGAT

AAGTACTACGCCGCTGATTATGACGAAGCTACAGGCGCCATTAAAGCTAAAACCACATCT

TATACCGCTGCCGATGGTACTACCAAGACTGCTGCCAATCAGCTGGGTGGAGTCGATGGA

AAAACCGAAGTGGTCACAATCGACGGAAAGACCTATCAAGCCTCCAAGGCTGCTGGCCAC

GACTTCAAGGCCCAACCCGAACTGGCCGAGGCTGCTGCTAAAACCACTGAAAACCCCCTG

CAGAAAATTGACGCTGCCCTGGCCCAAGTGGATGCCCTCCGCTCCGACCTCGGCGCTGTG

CAAAACCGCTTCAACTCCGCTATTACAAACCTCGGCAATACCGTCAATCAGCTCTCTGAA

GCTAGGTCTCGTATTGAGGATTCCGATTACGCTACCGAGGTCTCCCAGATGTCCCGTGCC

CAAATTCTCCAACAAGCCGGCACCTCCGTCCTCGCCCAAGCCAATCAGGTGCCACAGAAT

GTGCTGAGC

SEQ ID NO:99 bee variety toxin (mellitin)

ATGAAATTCTTAGTCAACGTTGCCCTTGTTTTTATGGTCGTATACATTTCTTACATCTAT

GCG

SEQ ID NO：100 STF2.HA0s

ATGAAATTCTTAGTCAACGTTGCCCTTGTTTTTATGGTCGTATACATTTCTTACATCTAT

GCGGCCCAGGTTATCAATACCAACTCCCTGTCGTTGCTCACCCAAAATAACCTTAATAAA

AGCCAGAGCGCACTGGGAACCGCCATAGAACGCCTCTCAAGCGGCCTCCGGATCAATTCT

GCAAAAGACGACGCCGCCGGTCAGGCCATCGCAAACCGCTTTACCGCCAATATCAAGGGA

CTGACGCAGGCTTCGAGGAATGCTAACGATGGAATAAGCATCGCTCAAACCACGGAGGGC

GCCCTGAACGAGATCAACAACAACCTACAGCGCGTCAGGGAGCTCGCAGTGCAGTCCGCC

AATTCGACCAACTCGCAGTCGGACCTGGACTCGATCCAAGCCGAAATCACCCAGCGCCTG

AATGAGATTGACCGGGTGAGCGGTCAGACACAGTTTAACGGCGTGAAGGTACTTGCACAG

GATAACACACTTACGATACAGGTGGGCGCCAACGATGGTGAAACCATAGACATTGATCTC

AAACAGATTAACAGCCAGACGCTCGGGTTGGATAGCCTGAATGTGCAAAAGGCGTACGAC

GTGAAAGACACGGCGGTCACTACCAAAGCCTACGCTAACAATGGCACTACCTTGGATGTG

AGCGGATTGGATGATGCAGCAATCAAGGCTGCTACCGGCGGTACGAACGGAACCGCGTCC

GTGACCGGCGGTGCCGTGAAGTTCGATGCTGACAACAATAAGTATTTCGTCACCATTGGA

GGCTTTACTGGCGCCGACGCAGCAAAGAACGGCGACTATGAAGTGAACGTGGCAACCGAT

GGAACCGTGACGCTGGCCGCTGGTGCCACCAAGACCACCATGCCAGCCGGCGCCACAACT

AAGACCGAGGTGCAGGAGTTAAAGGACACCCCCGCGGTGGTTAGCGCAGATGCCAAAAAC

GCCTTGATCGCCGGCGGAGTGGATGCAACTGATGCTAATGGTGCGGAGCTGGTTAAAATG

TCGTATACAGACAAGAATGGTAAGACGATCGAGGGCGGTTATGCCCTTAAGGCAGGAGAT

AAGTATTACGCTGCTGATTACGATGAGGCGACGGGAGCTATTAAGGCCAAGACAACGTCA

TACACGGCGGCGGACGGAACGACTAAGACGGCTGCCAATCAGTTGGGAGGGGTTGACGGG

AAGACAGAGGTCGTTACGATCGATGGCAAGACATACAACGCTTCCAAGGCCGCTGGCCAC

GATTTCAAAGCTCAACCCGAACTGGCCGAGGCCGCGGCGAAAACAACTGAGAACCCGTTG

CAGAAGATTGATGCGGCCCTGGCGCAAGTAGATGCCCTGCGCTCAGACCTGGGCGCCGTT

CAAAATCGATTCAATTCCGCGATTACAAACCTGGGCAATACAGTAAACAATCTATCCGAG

GCCAGATCCCGCATTGAAGACTCCGACTACGCGACAGAAGTAAGTAACATGAGTCGTGCC

CAGATTCTGCAGCAGGCCGGCACTAGTGTCCTGGCCCAGGCCAATCAAGTCCCGCAGAAT

GTGCTGAGCCTACTAGCATCAGGTTCCGGCTCAGGTTCCGACACCATTTGCATTGGATAC

CATGCAAACAACTCAACCGATACTGTTGATACCGTCCTTGAGAAGAACGTTACCGTCACG

CACTCGGTCAACCTATTAGAGGATAGCCACAACGGAAAGCTGTGCCGTCTGAAAGGCATA

GCGCCACTGCAGCTCGGCAAATGTAACATCGCAGGTTGGCTCCTTGGTAACCCGGAGTGC

GACCCCCTCCTCCCTGTACGATCCTGGAGTTATATCGTGGAGACCCCCAATAGCGAGAAC

GGAATTTGCTACCCAGGAGATTTCATAGACTACGAGGAGTTGCGCGAGCAGCTTTCGTCT

GTGAGCAGCTTCGAAAGGTTCGAGATATTCCCGAAGGAGAGCAGCTGGCCGAATCATAAC

ACTAACGGTGTGACAGCCGCCTGCAGTCATGAAGGAAAGAGTTCATTCTATCGCAACCTG

CTGTGGTTGACGGAGAAAGAGGGCAGCTACCCTAAGTTGAAGAACTCCTATGTGAACAAA

AAAGGCAAGGAGGTTCTGGTGCTGTGGGGCATACACCACCCCCCCAATAGCAAGGAGCAG

CAGAATCTGTACCAAAACGAGAATGCCTATGTGAGCGTGGTCACTAGTAACTATAACCGT

CGGTTCACTCCCGAGATCGCCGAGCGTCCGAAGGTGAGGGACCAGGCAGGCCGGATGAAC

TACTACTGGACCCTATTGAAGCCAGGGGACACGATTATCTTCGAGGCAAACGGAAACCTC

ATAGCGCCGATGTACGCCTTCGCCCTGAGCCGTGGCTTTGGATCGGGGATCATCACGTCT

AACGCCTCGATGCACGAATGTAATACCAAATGCCAGACCCCACTGGGTGCTATCAACTCG

TCCTTACCCTATCAAAATATACATCCGGTCACCATAGGCGAGTGTCCCAAATATGTCAGA

TCCGCCAAGTTGCGGATGGTGACCGGCCTCCGCAATATTCCTAGTATTCAGTCACGCGGC

TTGTTCGGCGCCATCGCTGGTTTCATCGAAGGCGGGTGGACAGGCATGATTGATGGCTGG

TATGGCTATCACCACCAGAACGAGCAGGGCTCGGGCTACGCGGCTGACCAGAAGTCGACT

CAGAATGCCATCAATGGCATCACGAATAAGGTGAACACGGTCATTGAAAAGATGAACATT

CAATTTACAGCCGTAGGAAAAGAGTTTAATAAACTGGAAAAAAGAATGGAGAATCTGAAT

AAGAAGGTGGACGACGGATTTTTGGACATCTGGACGTACAACGCCGAGCTGCTGGTTCTG

CTGGAAAATGAGCGAACACTGGATTTTCATGATTCTAACGTAAAGAATCTGTACGAGAAG

GTGAAGTCCCAACTAAAGAATAATGCCAAGGAAATCGGAAATGGATGCTTTGAGTTTTAC

CACAAGTGCGATAATGAGTGCATGGAATCCGTGCGAAATGGTACATACGATTACCCAAAG

TACTCCGAAGAATCCAAGCTAAATCGCGAAAAGGTTGATGGTGTTAAACTTGAATCCATG

GGTATTTACCAACACCATCATCACCACCATTAATAG

SEQ ID NO:101 FOR primer

AGTCGCTGAGCCTACTAGCATCAGGTTCCGGCTCAGGTTCCGACACCATTTGCATTGGAT

ACCATGC

SEQ ID NO:102 REV primer

AGTCGCATGCCTATTAATGGTGGTGATGATGGTGTTGGTAAATACCCATGGATTC

SEQ ID NO：103 HA0s PR8

GACACCATTTGCATTGGATACCATGCAAACAACTCAACCGATACTGTTGATACCGTCCTT

GAGAAGAACGTTACCGTCACGCACTCGGTCAACCTATTAGAGGATAGCCACAACGGAAAG

CTGTGCCGTCTGAAAGGCATAGCGCCACTGCAGCTCGGCAAATGTAACATCGCAGGTTGG

CTCCTTGGTAACCCGGAGTGCGACCCCCTCCTCCCTGTACGATCCTGGAGTTATATCGTG

GAGACCCCCAATAGCGAGAACGGAATTTGCTACCCAGGAGATTTCATAGACTACGAGGAG

TTGCGCGAGCAGCTTTCGTCTGTGAGCAGCTTCGAAAGGTTCGAGATATTCCCGAAGGAG

AGCAGCTGGCCGAATCATAACACTAACGGTGTGACAGCCGCCTGCAGTCATGAAGGAAAG

AGTTCATTCTATCGCAACCTGCTGTGGTTGACGGAGAAAGAGGGCAGCTACCCTAAGTTG

AAGAACTCCTATGTGAACAAAAAAGGCAAGGAGGTTCTGGTGCTGTGGGGCATACACCAC

CCCCCCAATAGCAAGGAGCAGCAGAATCTGTACCAAAACGAGAATGCCTATGTGAGCGTG

GTCACTAGTAACTATAACCGTCGGTTCACTCCCGAGATCGCCGAGCGTCCGAAGGTGAGG

GACCAGGCAGGCCGGATGAACTACTACTGGACCCTATTGAAGCCAGGGGACACGATTATC

TTCGAGGCAAACGGAAACCTCATAGCGCCGATGTACGCCTTCGCCCTGAGCCGTGGCTTT

GGATCGGGGATCATCACGTCTAACGCCTCGATGCACGAATGTAATACCAAATGCCAGACC

CCACTGGGTGCTATCAACTCGTCCTTACCCTATCAAAATATACATCCGGTCACCATAGGC

GAGTGTCCCAAATATGTCAGATCCGCCAAGTTGCGGATGGTGACCGGCCTCCGCAATATT

CCTAGTATTCAGTCACGCGGCTTGTTCGGCGCCATCGCTGGTTTCATCGAAGGCGGGTGG

ACAGGCATGATTGATGGCTGGTATGGCTATCACCACCAGAACGAGCAGGGCTCGGGCTAC

GCGGCTGACCAGAAGTCGACTCAGAATGCCATCAATGGCATCACGAATAAGGTGAACACG

GTCATTGAAAAGATGAACATTCAATTTACAGCCGTAGGAAAAGAGTTTAATAAACTGGAA

AAAAGAATGGAGAATCTGAATAAGAAGGTGGACGACGGATTTTTGGACATCTGGACGTAC

AACGCCGAGCTGCTGGTTCTGCTGGAAAATGAGCGAACACTGGATTTTCATGATTCTAAC

GTAAAGAATCTGTACGAGAAGGTGAAGTCCCAACTAAAGAATAATGCCAAGGAAATCGGA

AATGGATGCTTTGAGTTTTACCACAAGTGCGATAATGAGTGCATGGAATCCGTGCGAAAT

GGTACATACGATTACCCAAAGTACTCCGAAGAATCCAAGCTAAATCGCGAAAAGGTTGAT

GGTGTTAAACTTGAATCCATGGGTATTTACCAACACCATCATCACCACCATTAATAG

SEQ ID NO：104 STF2.HA1-1

ATGAAATTCTTAGTCAACGTTGCCCTTGTTTTTATGGTCGTATACATTTCTTACATCTAT

GCGGCCCAGGTTATCAATACCAACTCCCTGTCGTTGCTCACCCAAAATAACCTTAATAAA

AGCCAGAGCGCACTGGGAACCGCCATAGAACGCCTCTCAAGCGGCCTCCGGATCAATTCT

GCAAAAGACGACGCCGCCGGTCAGGCCATCGCAAACCGCTTTACCGCCAATATCAAGGGA

CTGACGCAGGCTTCGAGGAATGCTAACGATGGAATAAGCATCGCTCAAACCACGGAGGGC

GCCCTGAACGAGATCAACAACAACCTACAGCGCGTCAGGGAGCTCGCAGTGCAGTCCGCC

AATTCGACCAACTCGCAGTCGGACCTGGACTCGATCCAAGCCGAAATCACCCAGCGCCTG

AATGAGATTGACCGGGTGAGCGGTCAGACACAGTTTAACGGCGTGAAGGTACTTGCACAG

GATAACACACTTACGATACAGGTGGGCGCCAACGATGGTGAAACCATAGACATTGATCTC

AAACAGATTAACAGCCAGACGCTCGGGTTGGATAGCCTGAATGTGCAAAAGGCGTACGAC

GTGAAAGACACGGCGGTCACTACCAAAGCCTACGCTAACAATGGCACTACCTTGGATGTG

AGCGGATTGGATGATGCAGCAATCAAGGCTGCTACCGGCGGTACGAACGGAACCGCGTCC

GTGACCGGCGGTGCCGTGAAGTTCGATGCTGACAACAATAAGTATTTCGTCACCATTGGA

GGCTTTACTGGCGCCGACGCAGCAAAGAACGGCGACTATGAAGTGAACGTGGCAACCGAT

GGAACCGTGACGCTGGCCGCTGGTGCCACCAAGACCACCATGCCAGCCGGCGCCACAACT

AAGACCGAGGTGCAGGAGTTAAAGGACACCCCCGCGGTGGTTAGCGCAGATGCCAAAAAC

GCCTTGATCGCCGGCGGAGTGGATGCAACTGATGCTAATGGTGCGGAGCTGGTTAAAATG

TCGTATACAGACAAGAATGGTAAGACGATCGAGGGCGGTTATGCCCTTAAGGCAGGAGAT

AAGTATTACGCTGCTGATTACGATGAGGCGACGGGAGCTATTAAGGCCAAGACAACGTCA

TACACGGCGGCGGACGGAACGACTAAGACGGCTGCCAATCAGTTGGGAGGGGTTGACGGG

AAGACAGAGGTCGTTACGATCGATGGCAAGACATACAACGCTTCCAAGGCCGCTGGCCAC

GATTTCAAAGCTCAACCCGAACTGGCCGAGGCCGCGGCGAAAACAACTGAGAACCCGTTG

CAGAAGATTGATGCGGCCCTGGCGCAAGTAGATGCCCTGCGCTCAGACCTGGGCGCCGTT

CAAAATCGATTCAATTCCGCGATTACAAACCTGGGCAATACAGTAAACAATCTATCCGAG

GCCAGATCCCGCATTGAAGACTCCGACTACGCGACAGAAGTAAGTAACATGAGTCGTGCC

CAGATTCTGCAGCAGGCCGGCACTAGTGTCCTGGCCCAGGCCAATCAAGTCCCGCAGAAT

GTGCTGAGCCTACTAGCATCAGGTTCCGGCTCAGGTTCCAGCCACAACGGAAAGCTGTGC

CGTCTGAAAGGCATAGCGCCACTGCAGCTCGGCAAATGTAACATCGCAGGTTGGCTCCTT

GGTAACCCGGAGTGCGACCCCCTCCTCCCTGTACGATCCTGGAGTTATATCGTGGAGACC

CCCAATAGCGAGAACGGAATTTGCTACCCAGGAGATTTCATAGACTACGAGGAGTTGCGC

GAGCAGCTTTCGTCTGTGAGCAGCTTCGAAAGGTTCGAGATATTCCCGAAGGAGAGCAGC

TGGCCGAATCATAACACTAACGGTGTGACAGCCGCCTGCAGTCATGAAGGAAAGAGTTCA

TTCTATCGCAACCTGCTGTGGTTGACGGAGAAAGAGGGCAGCTACCCTAAGTTGAAGAAC

TCCTATGTGAACAAAAAAGGCAAGGAGGTTCTGGTGCTGTGGGGCATACACCACCCCCCC

AATAGCAAGGAGCAGCAGAATCTGTACCAAAACGAGAATGCCTATGTGAGCGTGGTCACT

AGTAACTATAACCGTCGGTTCACTCCCGAGATCGCCGAGCGTCCGAAGGTGAGGGACCAG

GCAGGCCGGATGAACTACTACTGGACCCTATTGAAGCCAGGGGACACGATTATCTTCGAG

GCAAACGGAAACCTCATAGCGCCGATGTACGCCTTCGCCCTGAGCCGTGGCTTTGGATCG

GGGATCATCACGTCTAACGCCTCGATGCACGAATGTAATACCAAATGCCAGACCCCACTG

GGTGCTATCAACTCGTCCTTACCCTATCAAAATATACATCCGGTCACCATAGGCGAGTGT

CCCAAATATGTCAGACACCATCATCACCACCATTAATAG

SEQ ID NO:105 FOR primer

AGTCGCTGAGCCTACTAGCATCAGGTTCCGGCTCAGGTTCCAGCCACAACGGAAAGCTGT

GCCGTC

SEQ ID NO:106 REV primer

AGTCGCATGCCTATTAATGGTGGTGATGATGGTGTCTGACATATTTGGGACACTCGCCTA

TGGTGAC

SEQ ID NO：107 STF2.HA1-2

ATGAAATTCTTAGTCAACGTTGCCCTTGTTTTTATGGTCGTATACATTTCTTACATCTAT

GCGGCCCAGGTTATCAATACCAACTCCCTGTCGTTGCTCACCCAAAATAACCTTAATAAA

AGCCAGAGCGCACTGGGAACCGCCATAGAACGCCTCTCAAGCGGCCTCCGGATCAATTCT

GCAAAAGACGACGCCGCCGGTCAGGCCATCGCAAACCGCTTTACCGCCAATATCAAGGGA

CTGACGCAGGCTTCGAGGAATGCTAACGATGGGATAAGCATCGCTCAAACCACGGAGGGC

GCCCTGAACGAGATCAACAACAACCTACAGCGCGTCAGGGAGCTCGCAGTGCAGTCCGCC

AATTCGACCAACTCGCAGTCGGACCTGGACTCGATCCAAGCCGAAATCACCCAGCGCCTG

AACGAGATTGACCGGGTGAGCGGTCAGACACAGTTTAACGGCGTGAAGGTACTTGCGCAG

GATAACACACTTACGATACAGGTGGGCGCCAACGATGGTGAAACCATAGACATTGATCTC

AAACAGATTAACAGCCAGACGCTCGGGTTGGATAGCCTGAATGTGCAAAAGGCGTACGAC

GTGAAAGACACGGCGGTCACTACCAAAGCCTACGCTAACAATGGCACTACCTTGGATGTG

AGCGGATTGGATGATGCAGCAATCAAGGCTGCTACCGGCGGTACGAACGGAACCGCGTCC

GTGACCGGCGGTGCCGTGAAGTTCGATGCTGACAACAATAAGTATTTCGTCACCATTGGA

GGCTTTACTGGCGCCGACGCAGCAAAGAACGGCGACTATGAAGTGAACGTGGCAACCGAT

GGAACCGTGACGCTGGCCGCTGGTGCCACCAAGACCACCATGCCAGCCGGCGCCACAACT

AAGACCGAGGTGCAGGAGTTAAAGGACACCCCCGCGGTGGTTAGCGCAGATGCCAAAAAC

GCGTTGATCGCCGGCGGAGTGGATGCAACTGATGCTAATGGTGCGGAGCTGGTTAAAATG

TCGTATACAGACAAGAATGGTAAGACGATCGAGGGCGGTTATGCCCTTAAGGCAGGAGAT

AAGTATTACGCTGCTGATTACGATGAGGCGACGGGAGCTATTAAGGCCAAGACAACGTCA

TACACGGCGGCGGACGGAACGACTAAGACGGCTGCCAATCAGTTGGGAGGGGTTGACGGG

AAGACAGAGGTCGTTACGATCGATGGCAAGACATACAACGCCTCCAAGGCCGCTGGCCAC

GATTTCAAAGCTCAACCCGAACTGGCCGAGGCCGCGGCGAAAACAACTGAGAACCCGTTG

CAGAAGATTGATGCGGCCCTGGCGCAAGTAGATGCCCTGCGCTCAGACCTGGGCGCCGTT

CAAAATCGATTCAATTCCGCGATTACAAACCTGGGCAATACAGTAAACAATCTATCCGAG

GCCAGATCCCGCATTGAAGACTCCGACTACGCGACAGAAGTAAGTAACATGAGTCGTGCC

CAGATTCTGCAGCAGGCCGGCACTAGTGTCCTGGCCCAGGCCAATCAAGTCCCGCAGAAT

GTGCTGAGCCTACTAGCATCAGGTTCCGGCTCAGGTTCCAAAGGCATAGCGCCACTGCAG

CTCGGCAAATGTAACATCGCAGGTTGGCTCCTTGGTAACCCGGAGTGCGACCCCCTCCTC

CCTGTACGATCCTGGAGTTATATCGTGGAGACCCCCAATAGCGAGAACGGAATTTGCTAC

CCAGGAGATTTCATAGACTACGAGGAGTTGCGCGAGCAGCTTTCGTCTGTGAGCAGCTTC

GAGAGGTTCGAAATCTTCCCGAAGGAGAGCAGCTGGCCGAATCATAACACTAACGGTGTG

ACAGCCGCCTGCAGTCATGAAGGAAAGAGTTCATTCTATCGCAACCTGCTGTGGTTGACG

GAGAAAGAGGGCAGCTACCCTAAGTTGAAGAACTCCTATGTGAACAAAAAAGGCAAGGAG

GTTCTGGTGCTGTGGGGCATACACCACCCCCCCAATAGCAAGGAGCAGCAGAATCTGTAC

CAAAACGAGAATGCCTATGTGAGCGTGGTCACTAGTAACTATAACCGTCGGTTCACTCCC

GAGATCGCCGAGCGTCCGAAGGTGAGGGACCAGGCAGGCCGGATGAACTACTACTGGACC

CTATTGAAGCCAGGGGACACGATTATCTTCGAGGCAAACGGAAACCTCATAGCGCCGATG

TACGCGTTCGCCCTGAGCCGTGGCTTTGGATCGGGGATCATCACGTCTCACCATCATCAC

CACCATTAATAG

SEQ ID NO:108 FOR primer

AGTCGCTGAGCCTACTAGCATCAGGTTCCGGCTCAGGTTCCAAAGGCATAGCGCCACTGC

AGCTCG

SEQ ID NO:109 REV primer

AGTCGCATGCCTATTAATGGTGGTGATGATGGTGAGACGTGATGATCCCCGATCCAAAGC

CACGGCTCAG

SEQ ID NO：110 STF2.HA1-2mut

ATGAAATTCTTAGTCAACGTTGCCCTTGTTTTTATGGTCGTATACATTTCTTACATCTAT

GCGGCCCAGGTTATCAATACCAACTCCCTGTCGTTGCTCACCCAAAATAACCTTAATAAA

AGCCAGAGCGCACTGGGAACCGCCATAGAACGCCTCTCAAGCGGCCTCCGGATCAATTCT

GCAAAAGACGACGCCGCCGGTCAGGCCATCGCAAACCGCTTTACCGCCAATATCAAGGGA

CTGACGCAGGCTTCGAGGAATGCTAACGATGGAATAAGCATCGCTCAAACCACGGAGGGC

GCCCTGAACGAGATCAACAACAACCTACAGCGCGTCAGGGAGCTCGCAGTGCAGTCCGCC

AATTCGACCAACTCGCAGTCGGACCTGGACTCGATCCAAGCCGAAATCACCCAGCGCCTG

AATGAGATTGACCGGGTGAGCGGTCAGACACAGTTTAACGGCGTGAAGGTACTTGCACAG

GATAACACACTTACGATACAGGTGGGCGCCAACGATGGTGAAACCATAGACATTGATCTC

AAACAGATTAACAGCCAGACGCTCGGGTTGGATAGCCTGAATGTGCAAAAGGCGTACGAC

GTGAAAGACACGGCGGTCACTACCAAAGCCTACGCTAACAATGGCACTACCTTGGATGTG

AGCGGATTGGATGATGCAGCAATCAAGGCTGCTACCGGCGGTACGAACGGAACCGCGTCC

GTGACCGGCGGTGCCGTGAAGTTCGATGCTGACAACAATAAGTATTTCGTCACCATTGGA

GGCTTTACTGGCGCCGACGCAGCAAAGAACGGCGACTATGAAGTGAACGTGGCAACCGAT

GGAACCGTGACGCTGGCCGCTGGTGCCACCAAGACCACCATGCCAGCCGGCGCCACAACT

AAGACCGAGGTGCAGGAGTTAAAGGACACCCCCGCGGTGGTTAGCGCAGATGCCAAAAAC

GCGTTGATCGCCGGCGGAGTGGATGCAACTGATGCTAATGGTGCGGAGCTGGTTAAAATG

TCGTATACAGACAAGAATGGTAAGACGATCGAGGGCGGTTATGCCCTTAAGGCAGGAGAT

AAGTATTACGCTGCTGATTACGATGAGGCGACGGGAGCTATTAAGGCCAAGACAACGTCA

TACACGGCGGCGGACGGAACGACTAAGACGGCTGCCAATCAGTTGGGAGGGGTTGACGGG

AAGACAGAGGTCGTTACGATCGATGGCAAGACATACAACGCCTCCAAGGCCGCTGGCCAC

GATTTCAAAGCTCAACCCGAACTGGCCGAGGCCGCGGCGAAAACAACTGAGAACCCGTTG

CAGAAGATTGATGCGGCCCTGGCGCAAGTAGATGCCCTGCGCTCAGACCTGGGCGCCGTT

CAAAATCGATTCAATTCCGCGATTACAAACCTGGGCAATACAGTAAACAATCTATCCGAG

GCCAGATCCCGCATTGAAGACTCCGACTACGCGACAGAAGTAAGTAACATGAGTCGTGCC

CAGATTCTGCAGCAGGCCGGCACTAGTGTCCTGGCCCAGGCCAATCAAGTCCCGCAGAAT

GTGCTGAGCCTGCTGGCTTCTGGATCTGGATCTGGTTCTAAAGGCGCTGCCCCTCTGCAA

CTCGGCAAGTGCAATATTGCGGGGTGGCTGTTGGGCAACCCAGAATGTGACCCCCTCCTG

CCCGTCCGTTCTTGGTCTGACATCGCTGAAACACCTAACTCCGAGAACGGCATCTGTTAC

CCGGGCGACTTCATTGACTACGAGGAGCTCCGCGAACAGCTGTCTTCTGTCTCTTCTTTC

GAACGTTTCGAAATTTTCCCAAAGGAATCCTCCTGGCCAAACCATAACACCAACGGAGTG

ACCGCCGCCTGTAGCCATGAGGGCAAGTCTTCTTTCTACCGTAATCTGCTGTGGCTGACT

GAAAAAGAGGGTTCTTATCCCAAACTGAAGAACTCTTATGTCAACAAGAAGGGCAAAGAG

GTCCTGGTGCTGTGGGGTATCCACCACCCCCCCAACTCCAAGGAGCAGCAGAATCTGTAC

CAAAACGAAAATGCTTACGTGTCTGTCGTGACATCTAACTACAACCGGCGCTTCACGCCC

GAAATCGCCGAGCGTCCCAAAGTGCGCGATCAGGCCGGAAGGATGAACTACTACTGGACC

CTGCTCAAACCCGGAGATACCATCATATTCGAAGCCAATGGCAATCTCATCGCTCCCATG

TATGCTTTCGCTCTCTCTCGCGGATTCGGTTCCGGCATAATCACTAGCCACCACCACCAC

CACCACTAATAG

SEQ ID NO：111 HA1-2mut

AAGGCGCTGCCCCTCTGCAACTCGGCAAGTGCAATATTGCGGGGTGGCTGTTGGGCAACC

CAGAATGTGACCCCCTCCTGCCCGTCCGTTCTTGGTCTGACATCGCTGAAACACCTAACT

CCGAGAACGGCATCTGTTACCCGGGCGACTTCATTGACTACGAGGAGCTCCGCGAACAGC

TGTCTTCTGTCTCTTCTTTCGAACGTTTCGAAATTTTCCCAAAGGAATCCTCCTGGCCAA

ACCATAACACCAACGGAGTGACCGCCGCCTGTAGCCATGAGGGCAAGTCTTCTTTCTACC

GTAATCTGCTGTGGCTGACTGAAAAAGAGGGTTCTTATCCCAAACTGAAGAACTCTTATG

TCAACAAGAAGGGCAAAGAGGTCCTGGTGCTGTGGGGTATCCACCACCCCCCCAACTCCA

AGGAGCAGCAGAATCTGTACCAAAACGAAAATGCTTACGTGTCTGTCGTGACATCTAACT

ACAACCGGCGCTTCACGCCCGAAATCGCCGAGCGTCCCAAAGTGCGCGATCAGGCCGGAA

GGATGAACTACTACTGGACCCTGCTCAAACCCGGAGATACCATCATATTCGAAGCCAATG

GCAATCTCATCGCTCCCATGTATGCTTTCGCTCTCTCTCGCGGATTCGGTTCCGGCATAA

TCACTAGCCACCACCACCACCACCACTAATAG

SEQ ID NO：112 STF2.HA1-3

ATGAAATTCTTAGTCAACGTTGCCCTTGTTTTTATGGTCGTATACATTTCTTACATCTAT

GCGGCCCAGGTTATCAATACCAACTCCCTGTCGTTGCTCACCCAAAATAACCTTAATAAA

AGCCAGAGCGCACTGGGAACCGCCATAGAACGCCTCTCAAGCGGCCTCCGGATCAATTCT

GCAAAAGACGACGCCGCCGGTCAGGCCATCGCAAACCGCTTTACCGCCAATATCAAGGGA

CTGACGCAGGCTTCGAGGAATGCTAACGATGGGATAAGCATCGCTCAAACCACGGAGGGC

GCCCTGAACGAGATCAACAACAACCTACAGCGCGTCAGGGAGCTCGCAGTGCAGTCCGCC

AATTCGACCAACTCGCAGTCGGACCTGGACTCGATCCAAGCCGAAATCACCCAGCGCCTG

AACGAGATTGACCGGGTGAGCGGTCAGACACAGTTTAACGGCGTGAAGGTACTTGCGCAG

GATAACACACTTACGATACAGGTGGGCGCCAACGATGGTGAAACCATAGACATTGATCTC

AAACAGATTAACAGCCAGACGCTCGGGTTGGATAGCCTGAATGTGCAAAAGGCGTACGAC

GTGAAAGACACGGCGGTCACTACCAAAGCCTACGCTAACAATGGCACTACCTTGGATGTG

AGCGGATTGGATGATGCAGCAATCAAGGCTGCTACCGGCGGTACGAACGGAACCGCGTCC

GTGACCGGCGGTGCCGTGAAGTTCGATGCTGACAACAATAAGTATTTCGTCACCATTGGA

GGCTTTACTGGCGCCGACGCAGCAAAGAACGGCGACTATGAAGTGAACGTGGCAACCGAT

GGAACCGTGACGCTGGCCGCTGGTGCCACCAAGACCACCATGCCAGCCGGCGCCACAACT

AAGACCGAGGTGCAGGAGTTAAAGGACACCCCCGCGGTGGTTAGCGCAGATGCCAAAAAC

GCGTTGATCGCCGGCGGAGTGGATGCAACTGATGCTAATGGTGCGGAGCTGGTTAAAATG

TCGTATACAGACAAGAATGGTAAGACGATCGAGGGCGGTTATGCCCTTAAGGCAGGAGAT

AAGTATTACGCTGCTGATTACGATGAGGCGACGGGAGCTATTAAGGCCAAGACAACGTCA

TACACGGCGGCGGACGGAACGACTAAGACGGCTGCCAATCAGTTGGGAGGGGTTGACGGG

AAGACAGAGGTCGTTACGATCGATGGCAAGACATACAACGCCTCCAAGGCCGCTGGCCAC

GATTTCAAAGCTCAACCCGAACTGGCCGAGGCCGCGGCGAAAACAACTGAGAACCCGTTG

CAGAAGATTGATGCGGCCCTGGCGCAAGTAGATGCCCTGCGCTCAGACCTGGGCGCCGTT

CAAAATCGATTCAATTCCGCGATTACAAACCTGGGCAATACAGTAAACAATCTATCCGAG

GCCAGATCCCGCATTGAAGACTCCGACTACGCGACAGAAGTAAGTAACATGAGTCGTGCC

CAGATTCTGCAGCAGGCCGGCACTAGTGTCCTGGCCCAGGCCAATCAAGTCCCGCAGAAT

GTGCTGAGCCTACTAGCATCAGGTTCCGGCTCAGGTTCCAATAGCGAGAACGGAATTTGC

TACCCAGGAGATTTCATAGACTACGAGGAGTTGCGCGAGCAGCTTTCGTCTGTGAGCAGC

TTCGAGAGGTTCGAAATCTTCCCGAAGGAGAGCAGCTGGCCGAATCATAACACTAACGGT

GTGACAGCCGCCTGCAGTCATGAAGGAAAGAGTTCATTCTATCGCAACCTGCTGTGGTTG

ACGGAGAAGGAGGGCAGCTACCCTAAGCTGAAGAACTCCTATGTGAACAAAAAAGGCAAG

GAGGTTCTGGTGCTGTGGGGCATACACCACCCCCCCAATAGCAAGGAGCAGCAGAATCTG

TACCAAAACGAGAATGCCTATGTGAGCGTGGTCACTAGTAACTATAACCGTCGGTTCACT

CCCGAGATCGCCGAGCGTCCGAAGGTGAGGGACCAGGCAGGCCGGATGAACTACTACTGG

ACCCTATTGAAGCCAGGGGACACGATTATCTTCGAGGCAAACGGAAACCTCATAGCGCCG

ATGTACGCCTTCGCCCTGAGCCGTGGCCACCATCATCACCACCATTAATAG

SEQ ID NO:113 FOR primer

AGTCGCTGAGCCTACTAGCATCAGGTTCCGGCTCAGGTTCCAATAGCGAGAACGGAATTT

GCTACC

SEQ ID NO:114 REV primer

AGTCGCATGCCTATTAATGGTGGTGATGATGGTGGCCACGGCTCAGGGCGAAGG

SEQ ID NO：115 STF2.HA1-3mut

ATGAAATTCTTAGTCAACGTTGCCCTTGTTTTTATGGTCGTATACATTTCTTACATCTAT

GCGGCCCAGGTTATCAATACCAACTCCCTGTCGTTGCTCACCCAAAATAACCTTAATAAA

AGCCAGAGCGCACTGGGAACCGCCATAGAACGCCTCTCAAGCGGCCTCCGGATCAATTCT

GCAAAAGACGACGCCGCCGGTCAGGCCATCGCAAACCGCTTTACCGCCAATATCAAGGGA

CTGACGCAGGCTTCGAGGAATGCTAACGATGGAATAAGCATCGCTCAAACCACGGAGGGC

GCCCTGAACGAGATCAACAACAACCTACAGCGCGTCAGGGAGCTCGCAGTGCAGTCCGCC

AATTCGACCAACTCGCAGTCGGACCTGGACTCGATCCAAGCCGAAATCACCCAGCGCCTG

AATGAGATTGACCGGGTGAGCGGTCAGACACAGTTTAACGGCGTGAAGGTACTTGCACAG

GATAACACACTTACGATACAGGTGGGCGCCAACGATGGTGAAACCATAGACATTGATCTC

AAACAGATTAACAGCCAGACGCTCGGGTTGGATAGCCTGAATGTGCAAAAGGCGTACGAC

GTGAAAGACACGGCGGTCACTACCAAAGCCTACGCTAACAATGGCACTACCTTGGATGTG

AGCGGATTGGATGATGCAGCAATCAAGGCTGCTACCGGCGGTACGAACGGAACCGCGTCC

GTGACCGGCGGTGCCGTGAAGTTCGATGCTGACAACAATAAGTATTTCGTCACCATTGGA

GGCTTTACTGGCGCCGACGCAGCAAAGAACGGCGACTATGAAGTGAACGTGGCAACCGAT

GGAACCGTGACGCTGGCCGCTGGTGCCACCAAGACCACCATGCCAGCCGGCGCCACAACT

AAGACCGAGGTGCAGGAGTTAAAGGACACCCCCGCGGTGGTTAGCGCAGATGCCAAAAAC

GCGTTGATCGCCGGCGGAGTGGATGCAACTGATGCTAATGGTGCGGAGCTGGTTAAAATG

TCGTATACAGACAAGAATGGTAAGACGATCGAGGGCGGTTATGCCCTTAAGGCAGGAGAT

AAGTATTACGCTGCTGATTACGATGAGGCGACGGGAGCTATTAAGGCCAAGACAACGTCA

TACACGGCGGCGGACGGAACGACTAAGACGGCTGCCAATCAGTTGGGAGGGGTTGACGGG

AAGACAGAGGTCGTTACGATCGATGGCAAGACATACAACGCCTCCAAGGCCGCTGGCCAC

GATTTCAAAGCTCAACCCGAACTGGCCGAGGCCGCGGCGAAAACAACTGAGAACCCGTTG

CAGAAGATTGATGCGGCCCTGGCGCAAGTAGATGCCCTGCGCTCAGACCTGGGCGCCGTT

CAAAATCGATTCAATTCCGCGATTACAAACCTGGGCAATACAGTAAACAATCTATCCGAG

GCCAGATCCCGCATTGAAGACTCCGACTACGCGACAGAAGTAAGTAACATGAGTCGTGCC

CAGATTCTGCAGCAGGCCGGCACTAGTGTCCTGGCCCAGGCCAATCAAGTCCCGCAGAAT

GTGCTGAGCCTGCTGGCTTCTGGATCTGGATCTGGTTCTAACTCCGAGAACGAAATCTGT

TACCCGGGCGACTTCATTGACAAAGAGGAGCTCCGCGAACAGCTGTCTTCTGTCTCTTCT

TTCGAACGTTTCGAAATTTTCCCAAAGGAATCCTCCTGGCCAAACCATAACACCAACGGA

GTGACCGCCGCCTGTAGCCATGAGGGCAAGTCTTCTTTCTACCGTAATCTGCTGTGGCTG

ACTGAAAAAGAGGGTTCTTATCCCAAACTGAAGAACTCTTATGTCAACAAGAAGGGCAAA

GAGGTCCTGGTGCTGTGGGGTATCCACCACCCCCCCAACTCCAAGGAGCAGCAGAATCTG

TACCAAAACGAAAATGCTTACGTGTCTGTCGTGACATCTAACTACAACCGGCGCTTCACG

CCCGAAATCGCCGAGCGTCCCAAAGTGCGCGATCAGGCCGGAAGGATGAACTACTACTGG

ACCCTGCTCAAACCCGGAGATACCATCATATTCGAAGCCAATGGCAATCTCATCGCTCCC

ATGTATGCTGCTGCTCTCTCTCGCGGACACCACCACCACCACCACTAATAG

SEQ ID NO：116 HA1-3mut PR8

AACTCCGAGAACGAAATCTGTTACCCGGGCGACTTCATTGACAAAGAGGAGCTCCGCGAA

CAGCTGTCTTCTGTCTCTTCTTTCGAACGTTTCGAAATTTTCCCAAAGGAATCCTCCTGG

CCAAACCATAACACCAACGGAGTGACCGCCGCCTGTAGCCATGAGGGCAAGTCTTCTTTC

TACCGTAATCTGCTGTGGCTGACTGAAAAAGAGGGTTCTTATCCCAAACTGAAGAACTCT

TATGTCAACAAGAAGGGCAAAGAGGTCCTGGTGCTGTGGGGTATCCACCACCCCCCCAAC

TCCAAGGAGCAGCAGAATCTGTACCAAAACGAAAATGCTTACGTGTCTGTCGTGACATCT

AACTACAACCGGCGCTTCACGCCCGAAATCGCCGAGCGTCCCAAAGTGCGCGATCAGGCC

GGAAGGATGAACTACTACTGGACCCTGCTCAAACCCGGAGATACCATCATATTCGAAGCC

AATGGCAATCTCATCGCTCCCATGTATGCTGCTGCTCTCTCTCGCGGACACCACCACCAC

CACCACTAATAG

SEQ ID NO：117 ngSTF2.HA0s

ATGAAATTCTTAGTCAACGTTGCCCTTGTTTTTATGGTCGTATACATTTCTTACATCTAT

GCGGCCCAAGTGATCAACACCAACTCCCTGTCCCTGCTCACTCAAAACAACCTCCAGAAA

TCTCAGTCTGCTCTCGGTACTGCTATCGAGCGTCTCTCCTCCGGCCTGAGGATCAACTCC

GCTAAAGATGACGCCGCCGGACAAGCTATCGCTAACCGCTTTACTGCCAACATTAAAGGC

CTGACCCAAGCCTCTAGGAACGCCAATGATGGCATTTCTATCGCTCAAACCACCGAAGGC

GCCCTGAACGAAATCAATAATAACCTGCAACGTGTCCGCGAACTCGCCGTCCAGTCCGCC

CAATCTACCAACTCTCAGTCTGACCTGGACTCTATCCAAGCTGAGATCACCCAAAGGCTC

AACGAGATCGATCGTGTGTCTGGTCAAACTCAGTTTAACGGCGTCAAAGTGCTGGCTCAA

GACAACACACTCACCATCCAAGTAGGAGCCAATGACGGCGAGACAATCGATATCGACCTG

AAGCAGATCAATTCTCAAACTCTGGGCCTGGACTCCCTGAACGTCCAAAAGGCTTACGAC

GTGAAGGACACCGCTGTGACAACCAAAGCCTATGCTAATCAAGGAACAACCCTGGACGTG

TCCGGACTCGATGACGCCGCTATCAAGGCCGCTACTGGCGGCACTCAGGGAACTGCCTCT

GTGACCGGAGGCGCTGTGAAGTTCGACGCCGATAACAACAAATACTTCGTCACTATTGGT

GGTTTCACTGGCGCTGACGCCGCTAAGAACGGTGACTACGAGGTCAACGTCGCCACTGAC

GGAACAGTGACACTGGCCGCTGGCGCTACCAAGACCACAATGCCTGCTGGTGCTACTACT

AAGACAGAGGTGCAGGAACTCAAGGACACCCCTGCCGTGGTGTCCGCCGATGCTAAGAAC

GCTCTCATTGCTGGTGGTGTCGACGCTACGGACGCCAACGGAGCCGAGCTTGTGAAAATG

TCCTACACCGACAAGAACGGAAAGACTATCGAGGGAGGTTACGCTCTGAAGGCTGGCGAT

AAGTACTACGCCGCTGATTATGACGAAGCTACAGGCGCCATTAAAGCTAAAACCACATCT

TATACCGCTGCCGATGGTACTACCAAGACTGCTGCCAATCAGCTGGGTGGAGTCGATGGA

AAAACCGAAGTGGTCACAATCGACGGAAAGACCTATCAAGCCTCCAAGGCTGCTGGCCAC

GACTTCAAGGCCCAACCCGAACTGGCCGAGGCTGCTGCTAAAACCACTGAAAACCCCCTG

CAGAAAATTGACGCTGCCCTGGCCCAAGTGGATGCCCTCCGCTCCGACCTCGGCGCTGTG

CAAAACCGCTTCAACTCCGCTATTACAAACCTCGGCAATACCGTCAATCAACTCTCTGAA

GCTAGGTCTCGTATTGAGGATTCCGATTACGCTACCGAGGTCTCCCAGATGTCCCGTGCC

CAAATTCTCCAACAAGCCGGCACCTCCGTCCTCGCCCAAGCCAATCAGGTGCCACAGAAT

GTGCTGAGCCTGCTGGCTTCTGGATCTGGATCTGGTTCTGACACCATCTGCATTGGCTAC

CATGCCCAGCAGTCTACAGATACAGTCGATACCGTCCTGGAGAAACAAGTCACCGTCACG

CACTCCGTGAACCTCCTGGAGGACTCCCATAATGGCAAATTGTGTCGGCTGAAAGGCATC

GCCCCTCTGCAACTCGGCAAGTGCAATATTGCGGGGTGGCTGTTGGGCAACCCAGAATGT

GACCCCCTCCTGCCCGTCCGTTCTTGGTCTTACATCGTGGAAACACCTAACTCCGAGAAC

GGCATCTGTTACCCGGGCGACTTCATTGACTACGAGGAGCTCCGCGAACAGCTGTCTTCT

GTCTCTTCTTTCGAACGTTTCGAAATTTTCCCAAAGGAATCCTCCTGGCCAAACCATAAC

ACCAACGGAGTGACCGCCGCCTGTAGCCATGAGGGCAAGTCTTCTTTCTACCGTAATCTG

CTGTGGCTGACTGAAAAAGAGGGTTCTTATCCCAAACTGAAGAACTCTTATGTCAACAAG

AAGGGCAAAGAGGTCCTGGTGCTGTGGGGTATCCACCACCCCCCCAACTCCAAGGAGCAG

CAGAATCTGTACCAAAACGAAAATGCTTACGTGTCTGTCGTGACATCTAACTACAACCGG

CGCTTCACGCCCGAAATCGCCGAGCGTCCCAAAGTGCGCGATCAGGCCGGAAGGATGAAC

TACTACTGGACCCTGCTCAAACCCGGAGATACCATCATATTCGAAGCCAATGGCAATCTC

ATCGCTCCCATGTATGCTTTCGCTCTCTCTCGCGGATTCGGTTCCGGCATAATCACTAGC

CAAGCCTCCATGCACGAGTGCAATACCAAGTGTCAAACTCCACTGGGAGCCATTCAAAGC

TCCCTGCCCTACCAAAACATCCATCCTGTGACCATTGGAGAGTGCCCTAAATACGTGCGC

AGCGCGAAACTGCGCATGGTGACCGGTCTCCGCAATATACCCTCCATACAATCGCGCGGC

CTGTTTGGTGCTATCGCTGGCTTTATCGAGGGAGGATGGACTGGAATGATCGACGGCTGG

TATGGCTATCATCATCAAAACGAACAGGGTTCCGGCTACGCCGCTGACCAAAAGTCCACT

CAAAACGCCATTAACGGTATTACAAACAAAGTAAACACCGTGATAGAGAAAATGAATATC

CAATTCACTGCCGTGGGCAAAGAGTTTAACAAGCTGGAGAAGCGCATGGAAAATCTGAAC

AAAAAAGTCGATGATGGCTTCCTCGACATCTGGACTTACAACGCCGAACTCCTCGTGCTG

CTCGAAAACGAGAGGACTCTGGACTTCCACGACTCCAACGTGAAGAACCTGTACGAGAAA

GTCAAATCCCAACTCAAGAACAACGCCAAAGAAATCGGCAACGGCTGCTTCGAGTTCTAC

CACAAATGTGATAACGAGTGTATGGAATCCGTACGGCAAGGCACTTACGACTACCCCAAA

TACAGCGAAGAGAGCAAATTGAACCGCGAGAAAGTGGACGGCGTGAAACTGGAGTCCATG

GGCATCTATCAGCACCACCACCACCACCACTAATAG

SEQ ID NO：118 ngHA0s

GACACCATCTGCATTGGCTACCATGCCCAGCAGTCTACAGATACAGTCGATACCGTCCTG

GAGAAACAAGTCACCGTCACGCACTCCGTGAACCTCCTGGAGGACTCCCATAATGGCAAA

TTGTGTCGGCTGAAAGGCATCGCCCCTCTGCAACTCGGCAAGTGCAATATTGCGGGGTGG

CTGTTGGGCAACCCAGAATGTGACCCCCTCCTGCCCGTCCGTTCTTGGTCTTACATCGTG

GAAACACCTAACTCCGAGAACGGCATCTGTTACCCGGGCGACTTCATTGACTACGAGGAG

CTCCGCGAACAGCTGTCTTCTGTCTCTTCTTTCGAACGTTTCGAAATTTTCCCAAAGGAA

TCCTCCTGGCCAAACCATAACACCAACGGAGTGACCGCCGCCTGTAGCCATGAGGGCAAG

TCTTCTTTCTACCGTAATCTGCTGTGGCTGACTGAAAAAGAGGGTTCTTATCCCAAACTG

AAGAACTCTTATGTCAACAAGAAGGGCAAAGAGGTCCTGGTGCTGTGGGGTATCCACCAC

CCCCCCAACTCCAAGGAGCAGCAGAATCTGTACCAAAACGAAAATGCTTACGTGTCTGTC

GTGACATCTAACTACAACCGGCGCTTCACGCCCGAAATCGCCGAGCGTCCCAAAGTGCGC

GATCAGGCCGGAAGGATGAACTACTACTGGACCCTGCTCAAACCCGGAGATACCATCATA

TTCGAAGCCAATGGCAATCTCATCGCTCCCATGTATGCTTTCGCTCTCTCTCGCGGATTC

GGTTCCGGCATAATCACTAGCCAAGCCTCCATGCACGAGTGCAATACCAAGTGTCAAACT

CCACTGGGAGCCATTCAAAGCTCCCTGCCCTACCAAAACATCCATCCTGTGACCATTGGA

GAGTGCCCTAAATACGTGCGCAGCGCGAAACTGCGCATGGTGACCGGTCTCCGCAATATA

CCCTCCATACAATCGCGCGGCCTGTTTGGTGCTATCGCTGGCTTTATCGAGGGAGGATGG

ACTGGAATGATCGACGGCTGGTATGGCTATCATCATCAAAACGAACAGGGTTCCGGCTAC

GCCGCTGACCAAAAGTCCACTCAAAACGCCATTAACGGTATTACAAACAAAGTAAACACC

GTGATAGAGAAAATGAATATCCAATTCACTGCCGTGGGCAAAGAGTTTAACAAGCTGGAG

AAGCGCATGGAAAATCTGAACAAAAAAGTCGATGATGGCTTCCTCGACATCTGGACTTAC

AACGCCGAACTCCTCGTGCTGCTCGAAAACGAGAGGACTCTGGACTTCCACGACTCCAAC

GTGAAGAACCTGTACGAGAAAGTCAAATCCCAACTCAAGAACAACGCCAAAGAAATCGGC

AACGGCTGCTTCGAGTTCTACCACAAATGTGATAACGAGTGTATGGAATCCGTACGGCAA

GGCACTTACGACTACCCCAAATACAGCGAAGAGAGCAAATTGAACCGCGAGAAAGTGGAC

GGCGTGAAACTGGAGTCCATGGGCATCTATCAGCACCACCACCACCACCACTAATAG

SEQ ID NO：119 ngSTF2.HA1-1

ATGAAATTCTTAGTCAACGTTGCCCTTGTTTTTATGGTCGTATACATTTCTTACATCTAT

GCGGCCCAAGTGATCAACACCAACTCCCTGTCCCTGCTCACTCAAAACAACCTCCAGAAA

TCTCAGTCTGCTCTCGGTACTGCTATCGAGCGTCTCTCCTCCGGCCTGAGGATCAACTCC

GCTAAAGATGACGCCGCCGGACAAGCTATCGCTAACCGCTTTACTGCCAACATTAAAGGC

CTGACCCAAGCCTCTAGGAACGCCAATGATGGCATTTCTATCGCTCAAACCACCGAAGGC

GCCCTGAACGAAATCAATAATAACCTGCAACGTGTCCGCGAACTCGCCGTCCAGTCCGCC

CAATCTACCAACTCTCAGTCTGACCTGGACTCTATCCAAGCTGAGATCACCCAAAGGCTC

AACGAGATCGATCGTGTGTCTGGTCAAACTCAGTTTAACGGCGTCAAAGTGCTGGCTCAA

GACAACACACTCACCATCCAAGTAGGAGCCAATGACGGCGAGACAATCGATATCGACCTG

AAGCAGATCAATTCTCAAACTCTGGGCCTGGACTCCCTGAACGTCCAAAAGGCTTACGAC

GTGAAGGACACCGCTGTGACAACCAAAGCCTATGCTAATCAAGGAACAACCCTGGACGTG

TCCGGACTCGATGACGCCGCTATCAAGGCCGCTACTGGCGGCACTCAGGGAACTGCCTCT

GTGACCGGAGGCGCTGTGAAGTTCGACGCCGATAACAACAAATACTTCGTCACTATTGGT

GGTTTCACTGGCGCTGACGCCGCTAAGAACGGTGACTACGAGGTCAACGTCGCCACTGAC

GGAACAGTGACACTGGCCGCTGGCGCTACCAAGACCACAATGCCTGCTGGTGCTACTACT

AAGACAGAGGTGCAGGAACTCAAGGACACCCCTGCCGTGGTGTCCGCCGATGCTAAGAAC

GCTCTCATTGCTGGTGGTGTCGACGCTACGGACGCCAACGGAGCCGAGCTTGTGAAAATG

TCCTACACCGACAAGAACGGAAAGACTATCGAGGGAGGTTACGCTCTGAAGGCTGGCGAT

AAGTACTACGCCGCTGATTATGACGAAGCTACAGGCGCCATTAAAGCTAAAACCACATCT

TATACCGCTGCCGATGGTACTACCAAGACTGCTGCCAATCAGCTGGGTGGAGTCGATGGA

AAAACCGAAGTGGTCACAATCGACGGAAAGACCTATCAAGCCTCCAAGGCTGCTGGCCAC

GACTTCAAGGCCCAACCCGAACTGGCCGAGGCTGCTGCTAAAACCACTGAAAACCCCCTG

CAGAAAATTGACGCTGCCCTGGCCCAAGTGGATGCCCTCCGCTCCGACCTCGGCGCTGTG

CAAAACCGCTTCAACTCCGCTATTACAAACCTCGGCAATACCGTCAATCAGCTCTCTGAA

GCTAGGTCTCGTATTGAGGATTCCGATTACGCTACCGAGGTCTCCCAGATGTCCCGTGCC

CAAATTCTCCAACAAGCCGGCACCTCCGTCCTCGCCCAAGCCAATCAGGTGCCACAGAAT

GTGCTGAGCCTGCTGGCTTCTGGATCTGGATCTGGTTCTTCCCATAATGGCAAATTGTGT

CGGCTGAAAGGCATCGCCCCTCTGCAACTCGGCAAGTGCAATATTGCGGGGTGGCTGTTG

GGCAACCCAGAATGTGACCCCCTCCTGCCCGTCCGTTCTTGGTCTTACATCGTGGAAACA

CCTAACTCCGAGAACGGCATCTGTTACCCGGGCGACTTCATTGACTACGAGGAGCTCCGC

GAACAGCTGTCTTCTGTCTCTTCTTTCGAACGTTTCGAAATTTTCCCAAAGGAATCCTCC

TGGCCAAACCATAACACCAACGGAGTGACCGCCGCCTGTAGCCATGAGGGCAAGTCTTCT

TTCTACCGTAATCTGCTGTGGCTGACTGAAAAAGAGGGTTCTTATCCCAAACTGAAGAAC

TCTTATGTCAACAAGAAGGGCAAAGAGGTCCTGGTGCTGTGGGGTATCCACCACCCCCCC

AACTCCAAGGAGCAGCAGAATCTGTACCAAAACGAAAATGCTTACGTGTCTGTCGTGACA

TCTAACTACAACCGGCGCTTCACGCCCGAAATCGCCGAGCGTCCCAAAGTGCGCGATCAG

GCCGGAAGGATGAACTACTACTGGACCCTGCTCAAACCCGGAGATACCATCATATTCGAA

GCCAATGGCAATCTCATCGCTCCCATGTATGCTTTCGCTCTCTCTCGCGGATTCGGTTCC

GGCATAATCACTAGCCAAGCCTCCATGCACGAGTGCAATACCAAGTGTCAAACTCCACTG

GGAGCCATTCAAAGCTCCCTGCCCTACCAAAACATCCATCCTGTGACCATTGGAGAGTGC

CCTAAATACGTGCGCCACCACCACCACCACCACTAATAG

SEQ ID NO：120 ng HA1-1

TCCCATAATGGCAAATTGTGTCGGCTGAAAGGCATCGCCCCTCTGCAACTCGGCAAGTGC

AATATTGCGGGGTGGCTGTTGGGCAACCCAGAATGTGACCCCCTCCTGCCCGTCCGTTCT

TGGTCTTACATCGTGGAAACACCTAACTCCGAGAACGGCATCTGTTACCCGGGCGACTTC

ATTGACTACGAGGAGCTCCGCGAACAGCTGTCTTCTGTCTCTTCTTTCGAACGTTTCGAA

ATTTTCCCAAAGGAATCCTCCTGGCCAAACCATAACACCAACGGAGTGACCGCCGCCTGT

AGCCATGAGGGCAAGTCTTCTTTCTACCGTAATCTGCTGTGGCTGACTGAAAAAGAGGGT

TCTTATCCCAAACTGAAGAACTCTTATGTCAACAAGAAGGGCAAAGAGGTCCTGGTGCTG

TGGGGTATCCACCACCCCCCCAACTCCAAGGAGCAGCAGAATCTGTACCAAAACGAAAAT

GCTTACGTGTCTGTCGTGACATCTAACTACAACCGGCGCTTCACGCCCGAAATCGCCGAG

CGTCCCAAAGTGCGCGATCAGGCCGGAAGGATGAACTACTACTGGACCCTGCTCAAACCC

GGAGATACCATCATATTCGAAGCCAATGGCAATCTCATCGCTCCCATGTATGCTTTCGCT

CTCTCTCGCGGATTCGGTTCCGGCATAATCACTAGCCAAGCCTCCATGCACGAGTGCAAT

ACCAAGTGTCAAACTCCACTGGGAGCCATTCAAAGCTCCCTGCCCTACCAAAACATCCAT

CCTGTGACCATTGGAGAGTGCCCTAAATACGTGCGCCACCACCACCACCACCACTAATAG

SEQ ID NO：121 ngSTF2.HA1-2

ATGAAATTCTTAGTCAACGTTGCCCTTGTTTTTATGGTCGTATACATTTCTTACATCTAT

GCGGCCCAAGTGATCAACACCAACTCCCTGTCCCTGCTCACTCAAAACAACCTCCAGAAA

TCTCAGTCTGCTCTCGGTACTGCTATCGAGCGTCTCTCCTCCGGCCTGAGGATCAACTCC

GCTAAAGATGACGCCGCCGGACAAGCTATCGCTAACCGCTTTACTGCCAACATTAAAGGC

CTGACCCAAGCCTCTAGGAACGCCAATGATGGCATTTCTATCGCTCAAACCACCGAAGGC

GCCCTGAACGAAATCAATAATAACCTGCAACGTGTCCGCGAACTCGCCGTCCAGTCCGCC

CAATCTACCAACTCTCAGTCTGACCTGGACTCTATCCAAGCTGAGATCACCCAAAGGCTC

AACGAGATCGATCGTGTGTCTGGTCAAACTCAGTTTAACGGCGTCAAAGTGCTGGCTCAA

GACAACACACTCACCATCCAAGTAGGAGCCAATGACGGCGAGACAATCGATATCGACCTG

AAGCAGATCAATTCTCAAACTCTGGGCCTGGACTCCCTGAACGTCCAAAAGGCTTACGAC

GTGAAGGACACCGCTGTGACAACCAAAGCCTATGCTAATCAAGGAACAACCCTGGACGTG

TCCGGACTCGATGACGCCGCTATCAAGGCCGCTACTGGCGGCACTCAGGGAACTGCCTCT

GTGACCGGAGGCGCTGTGAAGTTCGACGCCGATAACAACAAATACTTCGTCACTATTGGT

GGTTTCACTGGCGCTGACGCCGCTAAGAACGGTGACTACGAGGTCAACGTCGCCACTGAC

GGAACAGTGACACTGGCCGCTGGCGCTACCAAGACCACAATGCCTGCTGGTGCTACTACT

AAGACAGAGGTGCAGGAACTCAAGGACACCCCTGCCGTGGTGTCCGCCGATGCTAAGAAC

GCTCTCATTGCTGGTGGTGTCGACGCTACGGACGCCAACGGAGCCGAGCTTGTGAAAATG

TCCTACACCGACAAGAACGGAAAGACTATCGAGGGAGGTTACGCTCTGAAGGCTGGCGAT

AAGTACTACGCCGCTGATTATGACGAAGCTACAGGCGCCATTAAAGCTAAAACCACATCT

TATACCGCTGCCGATGGTACTACCAAGACTGCTGCCAATCAGCTGGGTGGAGTCGATGGA

AAAACCGAAGTGGTCACAATCGACGGAAAGACCTATCAAGCCTCCAAGGCTGCTGGCCAC

GACTTCAAGGCCCAACCCGAACTGGCCGAGGCTGCTGCTAAAACCACTGAAAACCCCCTG

CAGAAAATTGACGCTGCCCTGGCCCAAGTGGATGCCCTCCGCTCCGACCTCGGCGCTGTG

CAAAACCGCTTCAACTCCGCTATTACAAACCTCGGCAATACCGTCAATCAGCTCTCTGAA

GCTAGGTCTCGTATTGAGGATTCCGATTACGCTACCGAGGTCTCCCAGATGTCCCGTGCC

CAAATTCTCCAACAAGCCGGCACCTCCGTCCTCGCCCAAGCCAATCAGGTGCCACAGAAT

GTGCTGAGCCTACTAGCATCAGGTTCCGGCTCAGGTTCCAAAGGCATAGCGCCACTGCAG

CTCGGCAAATGTAACATCGCAGGTTGGCTCCTTGGTAACCCGGAGTGCGACCCCCTCCTC

CCTGTACGATCCTGGAGTTATATCGTGGAGACCCCCAATAGCGAGAACGGAATTTGCTAC

CCAGGAGATTTCATAGACTACGAGGAGTTGCGCGAGCAGCTTTCGTCTGTGAGCAGCTTC

GAGAGGTTCGAAATCTTCCCGAAGGAGAGCAGCTGGCCGAATCATAACACTAACGGTGTG

ACAGCCGCCTGCAGTCATGAAGGAAAGAGTTCATTCTATCGCAACCTGCTGTGGTTGACG

GAGAAAGAGGGCAGCTACCCTAAGTTGAAGAACTCCTATGTGAACAAAAAAGGCAAGGAG

GTTCTGGTGCTGTGGGGCATACACCACCCCCCCAATAGCAAGGAGCAGCAGAATCTGTAC

CAAAACGAGAATGCCTATGTGAGCGTGGTCACTAGTAACTATAACCGTCGGTTCACTCCC

GAGATCGCCGAGCGTCCGAAGGTGAGGGACCAGGCAGGCCGGATGAACTACTACTGGACC

CTATTGAAGCCAGGGGACACGATTATCTTCGAGGCAAACGGAAACCTCATAGCGCCGATG

TACGCGTTCGCCCTGAGCCGTGGCTTTGGATCGGGGATCATCACGTCTCACCATCATCAC

CACCATTAATAG

SEQ ID NO：122 ng HA1-2

AAAGGCATAGCGCCACTGCAGCTCGGCAAATGTAACATCGCAGGTTGGCTCCTTGGTAAC

CCGGAGTGCGACCCCCTCCTCCCTGTACGATCCTGGAGTTATATCGTGGAGACCCCCAAT

AGCGAGAACGGAATTTGCTACCCAGGAGATTTCATAGACTACGAGGAGTTGCGCGAGCAG

CTTTCGTCTGTGAGCAGCTTCGAAAGGTTCGAGATATTCCCGAAGGAGAGCAGCTGGCCG

AATCATAACACTAACGGTGTGACAGCCGCCTGCAGTCATGAAGGAAAGAGTTCATTCTAT

CGCAACCTGCTGTGGTTGACGGAGAAAGAGGGCAGCTACCCTAAGTTGAAGAACTCCTAT

GTGAACAAAAAAGGCAAGGAGGTTCTGGTGCTGTGGGGCATACACCACCCCCCCAATAGC

AAGGAGCAGCAGAATCTGTACCAAAACGAGAATGCCTATGTGAGCGTGGTCACTAGTAAC

TATAACCGTCGGTTCACTCCCGAGATCGCCGAGCGTCCGAAGGTGAGGGACCAGGCAGGC

CGGATGAACTACTACTGGACCCTATTGAAGCCAGGGGACACGATTATCTTCGAGGCAAAC

GGAAACCTCATAGCGCCGATGTACGCCTTCGCCCTGAGCCGTGGCTTTGGATCGGGGATC

ATCACGTCTCACCATCATCACCACCATTAATAG

SEQ ID NO：123 ngSTF2.HA1-2mut

ATGAAATTCTTAGTCAACGTTGCCCTTGTTTTTATGGTCGTATACATTTCTTACATCTAT

GCGGCCCAAGTGATCAACACCAACTCCCTGTCCCTGCTCACTCAAAACAACCTCCAGAAA

TCTCAGTCTGCTCTCGGTACTGCTATCGAGCGTCTCTCCTCCGGCCTGAGGATCAACTCC

GCTAAAGATGACGCCGCCGGACAAGCTATCGCTAACCGCTTTACTGCCAACATTAAAGGC

CTGACCCAAGCCTCTAGGAACGCCAATGATGGCATTTCTATCGCTCAAACCACCGAAGGC

GCCCTGAACGAAATCAATAATAACCTGCAACGTGTCCGCGAACTCGCCGTCCAGTCCGCC

CAATCTACCAACTCTCAGTCTGACCTGGACTCTATCCAAGCTGAGATCACCCAAAGGCTC

AACGAGATCGATCGTGTGTCTGGTCAAACTCAGTTTAACGGCGTCAAAGTGCTGGCTCAA

GACAACACACTCACCATCCAAGTAGGAGCCAATGACGGCGAGACAATCGATATCGACCTG

AAGCAGATCAATTCTCAAACTCTGGGCCTGGACTCCCTGAACGTCCAAAAGGCTTACGAC

GTGAAGGACACCGCTGTGACAACCAAAGCCTATGCTAATCAAGGAACAACCCTGGACGTG

TCCGGACTCGATGACGCCGCTATCAAGGCCGCTACTGGCGGCACTCAGGGAACTGCCTCT

GTGACCGGAGGCGCTGTGAAGTTCGACGCCGATAACAACAAATACTTCGTCACTATTGGT

GGTTTCACTGGCGCTGACGCCGCTAAGAACGGTGACTACGAGGTCAACGTCGCCACTGAC

GGAACAGTGACACTGGCCGCTGGCGCTACCAAGACCACAATGCCTGCTGGTGCTACTACT

AAGACAGAGGTGCAGGAACTCAAGGACACCCCTGCCGTGGTGTCCGCCGATGCTAAGAAC

GCTCTCATTGCTGGTGGTGTCGACGCTACGGACGCCAACGGAGCCGAGCTTGTGAAAATG

TCCTACACCGACAAGAACGGAAAGACTATCGAGGGAGGTTACGCTCTGAAGGCTGGCGAT

AAGTACTACGCCGCTGATTATGACGAAGCTACAGGCGCCATTAAAGCTAAAACCACATCT

TATACCGCTGCCGATGGTACTACCAAGACTGCTGCCAATCAGCTGGGTGGAGTCGATGGA

AAAACCGAAGTGGTCACAATCGACGGAAAGACCTATCAAGCCTCCAAGGCTGCTGGCCAC

GACTTCAAGGCCCAACCCGAACTGGCCGAGGCTGCTGCTAAAACCACTGAAAACCCCCTG

CAGAAAATTGACGCTGCCCTGGCCCAAGTGGATGCCCTCCGCTCCGACCTCGGCGCTGTG

CAAAACCGCTTCAACTCCGCTATTACAAACCTCGGCAATACCGTCAATCAGCTCTCTGAA

GCTAGGTCTCGTATTGAGGATTCCGATTACGCTACCGAGGTCTCCCAGATGTCCCGTGCC

CAAATTCTCCAACAAGCCGGCACCTCCGTCCTCGCCCAAGCCAATCAGGTGCCACAGAAT

GTGCTGAGCCTGCTGGCTTCTGGATCTGGATCTGGTTCTAAAGGCGCTGCCCCTCTGCAA

CTCGGCAAGTGCAATATTGCGGGGTGGCTGTTGGGCAACCCAGAATGTGACCCCCTCCTG

CCCGTCCGTTCTTGGTCTGACATCGCTGAAACACCTAACTCCGAGAACGGCATCTGTTAC

CCGGGCGACTTCATTGACTACGAGGAGCTCCGCGAACAGCTGTCTTCTGTCTCTTCTTTC

GAACGTTTCGAAATTTTCCCAAAGGAATCCTCCTGGCCAAACCATAACACCAACGGAGTG

ACCGCCGCCTGTAGCCATGAGGGCAAGTCTTCTTTCTACCGTAATCTGCTGTGGCTGACT

GAAAAAGAGGGTTCTTATCCCAAACTGAAGAACTCTTATGTCAACAAGAAGGGCAAAGAG

GTCCTGGTGCTGTGGGGTATCCACCACCCCCCCAACTCCAAGGAGCAGCAGAATCTGTAC

CAAAACGAAAATGCTTACGTGTCTGTCGTGACATCTAACTACAACCGGCGCTTCACGCCC

GAAATCGCCGAGCGTCCCAAAGTGCGCGATCAGGCCGGAAGGATGAACTACTACTGGACC

CTGCTCAAACCCGGAGATACCATCATATTCGAAGCCAATGGCAATCTCATCGCTCCCATGTATGCTTTCGCTCTCTCTCGCGGATTCGGTTCCGGCATAATCACTAGCCACCACCACCAC

CACCACTAATAG

SEQ ID NO：124 ngHA1-2mut

AAAGGCGCTGCCCCTCTGCAACTCGGCAAGTGCAATATTGCGGGGTGGCTGTTGGGCAAC

CCAGAATGTGACCCCCTCCTGCCCGTCCGTTCTTGGTCTGACATCGCTGAAACACCTAAC

TCCGAGAACGGCATCTGTTACCCGGGCGACTTCATTGACTACGAGGAGCTCCGCGAACAG

CTGTCTTCTGTCTCTTCTTTCGAACGTTTCGAAATTTTCCCAAAGGAATCCTCCTGGCCA

AACCATAACACCAACGGAGTGACCGCCGCCTGTAGCCATGAGGGCAAGTCTTCTTTCTAC

CGTAATCTGCTGTGGCTGACTGAAAAAGAGGGTTCTTATCCCAAACTGAAGAACTCTTAT

GTCAACAAGAAGGGCAAAGAGGTCCTGGTGCTGTGGGGTATCCACCACCCCCCCAACTCC

AAGGAGCAGCAGAATCTGTACCAAAACGAAAATGCTTACGTGTCTGTCGTGACATCTAAC

TACAACCGGCGCTTCACGCCCGAAATCGCCGAGCGTCCCAAAGTGCGCGATCAGGCCGGA

AGGATGAACTACTACTGGACCCTGCTCAAACCCGGAGATACCATCATATTCGAAGCCAAT

GGCAATCTCATCGCTCCCATGTATGCTTTCGCTCTCTCTCGCGGATTCGGTTCCGGCATA

ATCACTAGCCACCACCACCACCACCACTAATAG

SEQ ID NO：125 ngSTF2.HA1-3

ATGAAATTCTTAGTCAACGTTGCCCTTGTTTTTATGGTCGTATACATTTCTTACATCTAT

GCGGCCCAAGTGATCAACACCAACTCCCTGTCCCTGCTCACTCAAAACAACCTCCAGAAA

TCTCAGTCTGCTCTCGGTACTGCTATCGAGCGTCTCTCCTCCGGCCTGAGGATCAACTCC

GCTAAAGATGACGCCGCCGGACAAGCTATCGCTAACCGCTTTACTGCCAACATTAAAGGC

CTGACCCAAGCCTCTAGGAACGCCAATGATGGCATTTCTATCGCTCAAACCACCGAAGGC

GCCCTGAACGAAATCAATAATAACCTGCAACGTGTCCGCGAACTCGCCGTCCAGTCCGCC

CAATCTACCAACTCTCAGTCTGACCTGGACTCTATCCAAGCTGAGATCACCCAAAGGCTC

AACGAGATCGATCGTGTGTCTGGTCAAACTCAGTTTAACGGCGTCAAAGTGCTGGCTCAA

GACAACACACTCACCATCCAAGTAGGAGCCAATGACGGCGAGACAATCGATATCGACCTG

AAGCAGATCAATTCTCAAACTCTGGGCCTGGACTCCCTGAACGTCCAAAAGGCTTACGAC

GTGAAGGACACCGCTGTGACAACCAAAGCCTATGCTAATCAAGGAACAACCCTGGACGTG

TCCGGACTCGATGACGCCGCTATCAAGGCCGCTACTGGCGGCACTCAGGGAACTGCCTCT

GTGACCGGAGGCGCTGTGAAGTTCGACGCCGATAACAACAAATACTTCGTCACTATTGGT

GGTTTCACTGGCGCTGACGCCGCTAAGAACGGTGACTACGAGGTCAACGTCGCCACTGAC

GGAACAGTGACACTGGCCGCTGGCGCTACCAAGACCACAATGCCTGCTGGTGCTACTACT

AAGACAGAGGTGCAGGAACTCAAGGACACCCCTGCCGTGGTGTCCGCCGATGCTAAGAAC

GCTCTCATTGCTGGTGGTGTCGACGCTACGGACGCCAACGGAGCCGAGCTTGTGAAAATG

TCCTACACCGACAAGAACGGAAAGACTATCGAGGGAGGTTACGCTCTGAAGGCTGGCGAT

AAGTACTACGCCGCTGATTATGACGAAGCTACAGGCGCCATTAAAGCTAAAACCACATCT

TATACCGCTGCCGATGGTACTACCAAGACTGCTGCCAATCAGCTGGGTGGAGTCGATGGA

AAAACCGAAGTGGTCACAATCGACGGAAAGACCTATCAAGCCTCCAAGGCTGCTGGCCAC

GACTTCAAGGCCCAACCCGAACTGGCCGAGGCTGCTGCTAAAACCACTGAAAACCCCCTG

CAGAAAATTGACGCTGCCCTGGCCCAAGTGGATGCCCTCCGCTCCGACCTCGGCGCTGTG

CAAAACCGCTTCAACTCCGCTATTACAAACCTCGGCAATACCGTCAATCAGCTCTCTGAA

GCTAGGTCTCGTATTGAGGATTCCGATTACGCTACCGAGGTCTCCCAGATGTCCCGTGCC

CAAATTCTCCAACAAGCCGGCACCTCCGTCCTCGCCCAAGCCAATCAGGTGCCACAGAAT

GTGCTGAGCCTACTAGCATCAGGTTCCGGCTCAGGTTCCAATAGCGAGAACGGAATTTGC

TACCCAGGAGATTTCATAGACTACGAGGAGTTGCGCGAGCAGCTTTCGTCTGTGAGCAGC

TTCGAGAGGTTCGAAATCTTCCCGAAGGAGAGCAGCTGGCCGAATCATAACACTAACGGT

GTGACAGCCGCCTGCAGTCATGAAGGAAAGAGTTCATTCTATCGCAACCTGCTGTGGTTG

ACGGAGAAGGAGGGCAGCTACCCTAAGCTGAAGAACTCCTATGTGAACAAAAAAGGCAAG

GAGGTTCTGGTGCTGTGGGGCATACACCACCCCCCCAATAGCAAGGAGCAGCAGAATCTG

TACCAAAACGAGAATGCCTATGTGAGCGTGGTCACTAGTAACTATAACCGTCGGTTCACT

CCCGAGATCGCCGAGCGTCCGAAGGTGAGGGACCAGGCAGGCCGGATGAACTACTACTGG

ACCCTATTGAAGCCAGGGGACACGATTATCTTCGAGGCAAACGGAAACCTCATAGCGCCG

ATGTACGCCTTCGCCCTGAGCCGTGGCCACCATCATCACCACCATTAATAG

SEQ ID NO：126 ngHA1-3

AATAGCGAGAACGGAATTTGCTACCCAGGAGATTTCATAGACTACGAGGAGTTGCGCGAG

CAGCTTTCGTCTGTGAGCAGCTTCGAAAGGTTCGAGATATTCCCGAAGGAGAGCAGCTGG

CCGAATCATAACACTAACGGTGTGACAGCCGCCTGCAGTCATGAAGGAAAGAGTTCATTC

TATCGCAACCTGCTGTGGTTGACGGAGAAAGAGGGCAGCTACCCTAAGTTGAAGAACTCC

TATGTGAACAAAAAAGGCAAGGAGGTTCTGGTGCTGTGGGGCATACACCACCCCCCCAAT

AGCAAGGAGCAGCAGAATCTGTACCAAAACGAGAATGCCTATGTGAGCGTGGTCACTAGT

AACTATAACCGTCGGTTCACTCCCGAGATCGCCGAGCGTCCGAAGGTGAGGGACCAGGCA

GGCCGGATGAACTACTACTGGACCCTATTGAAGCCAGGGGACACGATTATCTTCGAGGCA

AACGGAAACCTCATAGCGCCGATGTACGCCTTCGCCCTGAGCCGTGGCCACCATCATCAC

CACCATTAATAG

SEQ ID NO：127 ngSTF2.HA1-3mut

ATGAAATTCTTAGTCAACGTTGCCCTTGTTTTTATGGTCGTATACATTTCTTACATCTAT

GCGGCCCAAGTGATCAACACCAACTCCCTGTCCCTGCTCACTCAAAACAACCTCCAGAAA

TCTCAGTCTGCTCTCGGTACTGCTATCGAGCGTCTCTCCTCCGGCCTGAGGATCAACTCC

GCTAAAGATGACGCCGCCGGACAAGCTATCGCTAACCGCTTTACTGCCAACATTAAAGGC

CTGACCCAAGCCTCTAGGAACGCCAATGATGGCATTTCTATCGCTCAAACCACCGAAGGC

GCCCTGAACGAAATCAATAATAACCTGCAACGTGTCCGCGAACTCGCCGTCCAGTCCGCC

CAATCTACCAACTCTCAGTCTGACCTGGACTCTATCCAAGCTGAGATCACCCAAAGGCTC

AACGAGATCGATCGTGTGTCTGGTCAAACTCAGTTTAACGGCGTCAAAGTGCTGGCTCAA

GACAACACACTCACCATCCAAGTAGGAGCCAATGACGGCGAGACAATCGATATCGACCTG

AAGCAGATCAATTCTCAAACTCTGGGCCTGGACTCCCTGAACGTCCAAAAGGCTTACGAC

GTGAAGGACACCGCTGTGACAACCAAAGCCTATGCTAATCAAGGAACAACCCTGGACGTG

TCCGGACTCGATGACGCCGCTATCAAGGCCGCTACTGGCGGCACTCAGGGAACTGCCTCT

GTGACCGGAGGCGCTGTGAAGTTCGACGCCGATAACAACAAATACTTCGTCACTATTGGT

GGTTTCACTGGCGCTGACGCCGCTAAGAACGGTGACTACGAGGTCAACGTCGCCACTGAC

GGAACAGTGACACTGGCCGCTGGCGCTACCAAGACCACAATGCCTGCTGGTGCTACTACT

AAGACAGAGGTGCAGGAACTCAAGGACACCCCTGCCGTGGTGTCCGCCGATGCTAAGAAC

GCTCTCATTGCTGGTGGTGTCGACGCTACGGACGCCAACGGAGCCGAGCTTGTGAAAATG

TCCTACACCGACAAGAACGGAAAGACTATCGAGGGAGGTTACGCTCTGAAGGCTGGCGAT

AAGTACTACGCCGCTGATTATGACGAAGCTACAGGCGCCATTAAAGCTAAAACCACATCT

TATACCGCTGCCGATGGTACTACCAAGACTGCTGCCAATCAGCTGGGTGGAGTCGATGGA

AAAACCGAAGTGGTCACAATCGACGGAAAGACCTATCAAGCCTCCAAGGCTGCTGGCCAC

GACTTCAAGGCCCAACCCGAACTGGCCGAGGCTGCTGCTAAAACCACTGAAAACCCCCTG

CAGAAAATTGACGCTGCCCTGGCCCAAGTGGATGCCCTCCGCTCCGACCTCGGCGCTGTG

CAAAACCGCTTCAACTCCGCTATTACAAACCTCGGCAATACCGTCAATCAGCTCTCTGAA

GCTAGGTCTCGTATTGAGGATTCCGATTACGCTACCGAGGTCTCCCAGATGTCCCGTGCC

CAAATTCTCCAACAAGCCGGCACCTCCGTCCTCGCCCAAGCCAATCAGGTGCCACAGAAT

GTGCTGAGCCTGCTGGCTTCTGGATCTGGATCTGGTTCTAACTCCGAGAACGAAATCTGT

TACCCGGGCGACTTCATTGACAAAGAGGAGCTCCGCGAACAGCTGTCTTCTGTCTCTTCT

TTCGAACGTTTCGAAATTTTCCCAAAGGAATCCTCCTGGCCAAACCATAACACCAACGGA

GTGACCGCCGCCTGTAGCCATGAGGGCAAGTCTTCTTTCTACCGTAATCTGCTGTGGCTG

ACTGAAAAAGAGGGTTCTTATCCCAAACTGAAGAACTCTTATGTCAACAAGAAGGGCAAA

GAGGTCCTGGTGCTGTGGGGTATCCACCACCCCCCCAACTCCAAGGAGCAGCAGAATCTG

TACCAAAACGAAAATGCTTACGTGTCTGTCGTGACATCTAACTACAACCGGCGCTTCACG

CCCGAAATCGCCGAGCGTCCCAAAGTGCGCGATCAGGCCGGAAGGATGAACTACTACTGG

ACCCTGCTCAAACCCGGAGATACCATCATATTCGAAGCCAATGGCAATCTCATCGCTCCC

ATGTATGCTGCTGCTCTCTCTCGCGGACACCACCACCACCACCACTAATAG

SEQ ID NO：128 ngHA1-3mut

AACTCCGAGAACGAAATCTGTTACCCGGGCGACTTCATTGACAAAGAGGAGCTCCGCGAA

CAGCTGTCTTCTGTCTCTTCTTTCGAACGTTTCGAAATTTTCCCAAAGGAATCCTCCTGG

CCAAACCATAACACCAACGGAGTGACCGCCGCCTGTAGCCATGAGGGCAAGTCTTCTTTC

TACCGTAATCTGCTGTGGCTGACTGAAAAAGAGGGTTCTTATCCCAAACTGAAGAACTCT

TATGTCAACAAGAAGGGCAAAGAGGTCCTGGTGCTGTGGGGTATCCACCACCCCCCCAAC

TCCAAGGAGCAGCAGAATCTGTACCAAAACGAAAATGCTTACGTGTCTGTCGTGACATCT

AACTACAACCGGCGCTTCACGCCCGAAATCGCCGAGCGTCCCAAAGTGCGCGATCAGGCC

GGAAGGATGAACTACTACTGGACCCTGCTCAAACCCGGAGATACCATCATATTCGAAGCC

AATGGCAATCTCATCGCTCCCATGTATGCTGCTGCTCTCTCTCGCGGACACCACCACCAC

CACCACTAATAG

SEQ ID NO：129 wtSTF2.HA1-1ng

ATGAAATTCTTAGTCAACGTTGCCCTTGTTTTTATGGTCGTATACATTTCTTACATCTAT

GCGGCCCAGGTTATCAATACCAACTCCCTGTCGTTGCTCACCCAAAATAACCTTAATAAA

AGCCAGAGCGCACTGGGAACCGCCATAGAACGCCTCTCAAGCGGCCTCCGGATCAATTCT

GCAAAAGACGACGCCGCCGGTCAGGCCATCGCAAACCGCTTTACCGCCAATATCAAGGGA

CTGACGCAGGCTTCGAGGAATGCTAACGATGGAATAAGCATCGCTCAAACCACGGAGGGC

GCCCTGAACGAGATCAACAACAACCTACAGCGCGTCAGGGAGCTCGCAGTGCAGTCCGCC

AATTCGACCAACTCGCAGTCGGACCTGGACTCGATCCAAGCCGAAATCACCCAGCGCCTG

AATGAGATTGACCGGGTGAGCGGTCAGACACAGTTTAACGGCGTGAAGGTACTTGCACAG

GATAACACACTTACGATACAGGTGGGCGCCAACGATGGTGAAACCATAGACATTGATCTC

AAACAGATTAACAGCCAGACGCTCGGGTTGGATAGCCTGAATGTGCAAAAGGCGTACGAC

GTGAAAGACACGGCGGTCACTACCAAAGCCTACGCTAACAATGGCACTACCTTGGATGTG

AGCGGATTGGATGATGCAGCAATCAAGGCTGCTACCGGCGGTACGAACGGAACCGCGTCC

GTGACCGGCGGTGCCGTGAAGTTCGATGCTGACAACAATAAGTATTTCGTCACCATTGGA

GGCTTTACTGGCGCCGACGCAGCAAAGAACGGCGACTATGAAGTGAACGTGGCAACCGAT

GGAACCGTGACGCTGGCCGCTGGTGCCACCAAGACCACCATGCCAGCCGGCGCCACAACT

AAGACCGAGGTGCAGGAGTTAAAGGACACCCCCGCGGTGGTTAGCGCAGATGCCAAAAAC

GCCTTGATCGCCGGCGGAGTGGATGCAACTGATGCTAATGGTGCGGAGCTGGTTAAAATG

TCGTATACAGACAAGAATGGTAAGACGATCGAGGGCGGTTATGCCCTTAAGGCAGGAGAT

AAGTATTACGCTGCTGATTACGATGAGGCGACGGGAGCTATTAAGGCCAAGACAACGTCA

TACACGGCGGCGGACGGAACGACTAAGACGGCTGCCAATCAGTTGGGAGGGGTTGACGGG

AAGACAGAGGTCGTTACGATCGATGGCAAGACATACAACGCTTCCAAGGCCGCTGGCCAC

GATTTCAAAGCTCAACCCGAACTGGCCGAGGCCGCGGCGAAAACAACTGAGAACCCGTTG

CAGAAGATTGATGCGGCCCTGGCGCAAGTAGATGCCCTGCGCTCAGACCTGGGCGCCGTT

CAAAATCGATTCAATTCCGCGATTACAAACCTGGGCAATACAGTAAACAATCTATCCGAG

GCCAGATCCCGCATTGAAGACTCCGACTACGCGACAGAAGTAAGTAACATGAGTCGTGCC

CAGATTCTGCAGCAGGCCGGCACTAGTGTCCTGGCCCAGGCCAATCAAGTCCCGCAGAAT

GTGCTGAGCCTACTAGCATCTGGATCTGGATCTGGTTCTTCCCATAATGGCAAATTGTGT

CGGCTGAAAGGCATCGCCCCTCTGCAACTCGGCAAGTGCAATATTGCGGGGTGGCTGTTG

GGCAACCCAGAATGTGACCCCCTCCTGCCCGTCCGTTCTTGGTCTTACATCGTGGAAACA

CCTAACTCCGAGAACGGCATCTGTTACCCGGGCGACTTCATTGACTACGAGGAGCTCCGC

GAACAGCTGTCTTCTGTCTCTTCTTTCGAACGTTTCGAAATTTTCCCAAAGGAATCCTCC

TGGCCAAACCATAACACCAACGGAGTGACCGCCGCCTGTAGCCATGAGGGCAAGTCTTCT

TTCTACCGTAATCTGCTGTGGCTGACTGAAAAAGAGGGTTCTTATCCCAAACTGAAGAAC

TCTTATGTCAACAAGAAGGGCAAAGAGGTCCTGGTGCTGTGGGGTATCCACCACCCCCCC

AACTCCAAGGAGCAGCAGAATCTGTACCAAAACGAAAATGCTTACGTGTCTGTCGTGACA

TCTAACTACAACCGGCGCTTCACGCCCGAAATCGCCGAGCGTCCCAAAGTGCGCGATCAG

GCCGGAAGGATGAACTACTACTGGACCCTGCTCAAACCCGGAGATACCATCATATTCGAA

GCCAATGGCAATCTCATCGCTCCCATGTATGCTTTCGCTCTCTCTCGCGGATTCGGTTCC

GGCATAATCACTAGCCAAGCCTCCATGCACGAGTGCAATACCAAGTGTCAAACTCCACTG

GGAGCCATTCAAAGCTCCCTGCCCTACCAAAACATCCATCCTGTGACCATTGGAGAGTGC

CCTAAATACGTGCGCCACCACCACCACCACCACTAATAG

SEQ ID NO：130 ngHA1-1

TCCCATAATGGCAAATTGTGTCGGCTGAAAGGCATCGCCCCTCTGCAACTCGGCAAGTGC

AATATTGCGGGGTGGCTGTTGGGCAACCCAGAATGTGACCCCCTCCTGCCCGTCCGTTCT

TGGTCTTACATCGTGGAAACACCTAACTCCGAGAACGGCATCTGTTACCCGGGCGACTTC

ATTGACTACGAGGAGCTCCGCGAACAGCTGTCTTCTGTCTCTTCTTTCGAACGTTTCGAA

ATTTTCCCAAAGGAATCCTCCTGGCCAAACCATAACACCAACGGAGTGACCGCCGCCTGT

AGCCATGAGGGCAAGTCTTCTTTCTACCGTAATCTGCTGTGGCTGACTGAAAAAGAGGGT

TCTTATCCCAAACTGAAGAACTCTTATGTCAACAAGAAGGGCAAAGAGGTCCTGGTGCTG

TGGGGTATCCACCACCCCCCCAACTCCAAGGAGCAGCAGAATCTGTACCAAAACGAAAAT

GCTTACGTGTCTGTCGTGACATCTAACTACAACCGGCGCTTCACGCCCGAAATCGCCGAG

CGTCCCAAAGTGCGCGATCAGGCCGGAAGGATGAACTACTACTGGACCCTGCTCAAACCC

GGAGATACCATCATATTCGAAGCCAATGGCAATCTCATCGCTCCCATGTATGCTTTCGCT

CTCTCTCGCGGATTCGGTTCCGGCATAATCACTAGCCAAGCCTCCATGCACGAGTGCAAT

ACCAAGTGTCAAACTCCACTGGGAGCCATTCAAAGCTCCCTGCCCTACCAAAACATCCAT

CCTGTGACCATTGGAGAGTGCCCTAAATACGTGCGCCACCACCACCACCACCACTAATAG

SEQ ID NO：131 ngSTF2.HA1-1wt

ATGAAATTCTTAGTCAACGTTGCCCTTGTTTTTATGGTCGTATACATTTCTTACATCTAT

GCGGCCCAAGTGATCAACACCAACTCCCTGTCCCTGCTCACTCAAAACAACCTCCAGAAA

TCTCAGTCTGCTCTCGGTACTGCTATCGAGCGTCTCTCCTCCGGCCTGAGGATCAACTCC

GCTAAAGATGACGCCGCCGGACAAGCTATCGCTAACCGCTTTACTGCCAACATTAAAGGC

CTGACCCAAGCCTCTAGGAACGCCAATGATGGCATTTCTATCGCTCAAACCACCGAAGGC

GCCCTGAACGAAATCAATAATAACCTGCAACGTGTCCGCGAACTCGCCGTCCAGTCCGCC

CAATCTACCAACTCTCAGTCTGACCTGGACTCTATCCAAGCTGAGATCACCCAAAGGCTC

AACGAGATCGATCGTGTGTCTGGTCAAACTCAGTTTAACGGCGTCAAAGTGCTGGCTCAA

GACAACACACTCACCATCCAAGTAGGAGCCAATGACGGCGAGACAATCGATATCGACCTG

AAGCAGATCAATTCTCAAACTCTGGGCCTGGACTCCCTGAACGTCCAAAAGGCTTACGAC

GTGAAGGACACCGCTGTGACAACCAAAGCCTATGCTAATCAAGGAACAACCCTGGACGTG

TCCGGACTCGATGACGCCGCTATCAAGGCCGCTACTGGCGGCACTCAGGGAACTGCCTCT

GTGACCGGAGGCGCTGTGAAGTTCGACGCCGATAACAACAAATACTTCGTCACTATTGGT

GGTTTCACTGGCGCTGACGCCGCTAAGAACGGTGACTACGAGGTCAACGTCGCCACTGAC

GGAACAGTGACACTGGCCGCTGGCGCTACCAAGACCACAATGCCTGCTGGTGCTACTACT

AAGACAGAGGTGCAGGAACTCAAGGACACCCCTGCCGTGGTGTCCGCCGATGCTAAGAAC

GCTCTCATTGCTGGTGGTGTCGACGCTACGGACGCCAACGGAGCCGAGCTTGTGAAAATG

TCCTACACCGACAAGAACGGAAAGACTATCGAGGGAGGTTACGCTCTGAAGGCTGGCGAT

AAGTACTACGCCGCTGATTATGACGAAGCTACAGGCGCCATTAAAGCTAAAACCACATCT

TATACCGCTGCCGATGGTACTACCAAGACTGCTGCCAATCAGCTGGGTGGAGTCGATGGA

AAAACCGAAGTGGTCACAATCGACGGAAAGACCTATCAAGCCTCCAAGGCTGCTGGCCAC

GACTTCAAGGCCCAACCCGAACTGGCCGAGGCTGCTGCTAAAACCACTGAAAACCCCCTG

CAGAAAATTGACGCTGCCCTGGCCCAAGTGGATGCCCTCCGCTCCGACCTCGGCGCTGTG

CAAAACCGCTTCAACTCCGCTATTACAAACCTCGGCAATACCGTCAATCAGCTCTCTGAA

GCTAGGTCTCGTATTGAGGATTCCGATTACGCTACCGAGGTCTCCCAGATGTCCCGTGCC

CAAATTCTCCAACAAGCCGGCACCTCCGTCCTCGCCCAAGCCAATCAGGTGCCACAGAAT

GTGCTGAGCCTACTAGCATCAGGTTCCGGCTCAGGTTCCAGCCACAACGGAAAGCTGTGC

CGTCTGAAAGGCATAGCGCCACTGCAGCTCGGCAAATGTAACATCGCAGGTTGGCTCCTT

GGTAACCCGGAGTGCGACCCCCTCCTCCCTGTACGATCCTGGAGTTATATCGTGGAGACC

CCCAATAGCGAGAACGGAATTTGCTACCCAGGAGATTTCATAGACTACGAGGAGTTGCGC

GAGCAGCTTTCGTCTGTGAGCAGCTTCGAGAGGTTCGAAATCTTCCCGAAGGAGAGCAGC

TGGCCGAATCATAACACTAACGGTGTGACAGCCGCCTGCAGTCATGAAGGAAAGAGTTCA

TTCTATCGCAACCTGCTGTGGTTGACGGAGAAAGAGGGCAGCTACCCTAAGTTGAAGAAC

TCCTATGTGAACAAAAAAGGCAAGGAGGTTCTGGTGCTGTGGGGCATACACCACCCCCCC

AATAGCAAGGAGCAGCAGAATCTGTACCAAAACGAGAATGCCTATGTGAGCGTGGTCACT

AGTAACTATAACCGTCGGTTCACTCCCGAGATCGCCGAGCGTCCGAAGGTGAGGGACCAG

GCAGGCCGGATGAACTACTACTGGACCCTATTGAAGCCAGGGGACACGATTATCTTCGAG

GCAAACGGAAACCTCATAGCGCCGATGTACGCGTTCGCCCTGAGCCGCGGCTTTGGATCG

GGGATCATCACGTCTAACGCCTCGATGCACGAATGTAATACCAAATGCCAGACCCCACTG

GGTGCTATCAACTCGTCCTTACCCTATCAAAATATACATCCGGTCACCATAGGCGAGTGT

CCCAAATATGTCAGACACCATCATCACCACCATTAATAG

SEQ ID NO：132 HA1-1 PR8

AGCCACAACGGAAAGCTGTGCCGTCTGAAAGGCATAGCGCCACTGCAGCTCGGCAAATGT

AACATCGCAGGTTGGCTCCTTGGTAACCCGGAGTGCGACCCCCTCCTCCCTGTACGATCC

TGGAGTTATATCGTGGAGACCCCCAATAGCGAGAACGGAATTTGCTACCCAGGAGATTTC

ATAGACTACGAGGAGTTGCGCGAGCAGCTTTCGTCTGTGAGCAGCTTCGAAAGGTTCGAG

ATATTCCCGAAGGAGAGCAGCTGGCCGAATCATAACACTAACGGTGTGACAGCCGCCTGC

AGTCATGAAGGAAAGAGTTCATTCTATCGCAACCTGCTGTGGTTGACGGAGAAAGAGGGC

AGCTACCCTAAGTTGAAGAACTCCTATGTGAACAAAAAAGGCAAGGAGGTTCTGGTGCTG

TGGGGCATACACCACCCCCCCAATAGCAAGGAGCAGCAGAATCTGTACCAAAACGAGAAT

GCCTATGTGAGCGTGGTCACTAGTAACTATAACCGTCGGTTCACTCCCGAGATCGCCGAG

CGTCCGAAGGTGAGGGACCAGGCAGGCCGGATGAACTACTACTGGACCCTATTGAAGCCA

GGGGACACGATTATCTTCGAGGCAAACGGAAACCTCATAGCGCCGATGTACGCCTTCGCC

CTGAGCCGTGGCTTTGGATCGGGGATCATCACGTCTAACGCCTCGATGCACGAATGTAAT

ACCAAATGCCAGACCCCACTGGGTGCTATCAACTCGTCCTTACCCTATCAAAATATACAT

CCGGTCACCATAGGCGAGTGTCCCAAATATGTCAGACACCATCATCACCACCATTAATAG

SEQ ID NO：133 HA1-1 PR8

ATGAAATTCTTAGTCAACGTTGCCCTTGTTTTTATGGTCGTATACATTTCTTACATCTAT

GCGAGCCACAACGGAAAGCTGTGCCGTCTGAAAGGCATAGCGCCACTGCAGCTCGGCAAA

TGTAACATCGCAGGTTGGCTCCTTGGTAACCCGGAGTGCGACCCCCTCCTCCCTGTACGA

TCCTGGAGTTATATCGTGGAGACCCCCAATAGCGAGAACGGAATTTGCTACCCAGGAGAT

TTCATAGACTACGAGGAGTTGCGCGAGCAGCTTTCGTCTGTGAGCAGCTTCGAAAGGTTC

GAGATATTCCCGAAGGAGAGCAGCTGGCCGAATCATAACACTAACGGTGTGACAGCCGCC

TGCAGTCATGAAGGAAAGAGTTCATTCTATCGCAACCTGCTGTGGTTGACGGAGAAAGAG

GGCAGCTACCCTAAGTTGAAGAACTCCTATGTGAACAAAAAAGGCAAGGAGGTTCTGGTG

CTGTGGGGCATACACCACCCCCCCAATAGCAAGGAGCAGCAGAATCTGTACCAAAACGAG

AATGCCTATGTGAGCGTGGTCACTAGTAACTATAACCGTCGGTTCACTCCCGAGATCGCC

GAGCGTCCGAAGGTGAGGGACCAGGCAGGCCGGATGAACTACTACTGGACCCTATTGAAG

CCAGGGGACACGATTATCTTCGAGGCAAACGGAAACCTCATAGCGCCGATGTACGCCTTC

GCCCTGAGCCGTGGCTTTGGATCGGGGATCATCACGTCTAACGCCTCGATGCACGAATGT

AATACCAAATGCCAGACCCCACTGGGTGCTATCAACTCGTCCTTACCCTATCAAAATATA

CATCCGGTCACCATAGGCGAGTGTCCCAAATATGTCAGACACCATCATCACCACCATTAAT

AG

SEQ ID NO:134 FOR primer

AGGCAGATCTATGAAATTCTTAGTCAACGTTGCCCTTGTTTTTATGGTCGTATACATTTC

TTACATCTATGCGAGCCACAACGGAAAGCTGTGCCGTCTGAAAGG

SEQ ID NO:135 REV primer

ACCTGCATGCCTATTAATGGTGGTGATGATGGTGTCTGACATATTTGGGACACTC

SEQ ID NO：136 HA0s PR8

ATGAAATTCTTAGTCAACGTTGCCCTTGTTTTTATGGTCGTATACATTTCTTACATCTAT

GCGGACACCATTTGCATTGGATACCATGCAAACAACTCAACCGATACTGTTGATACCGTC

CTTGAGAAGAACGTTACCGTCACGCACTCGGTCAACCTATTAGAGGATAGCCACAACGGA

AAGCTGTGCCGTCTGAAAGGCATAGCGCCACTGCAGCTCGGCAAATGTAACATCGCAGGT

TGGCTCCTTGGTAACCCGGAGTGCGACCCCCTCCTCCCTGTACGATCCTGGAGTTATATC

GTGGAGACCCCCAATAGCGAGAACGGAATTTGCTACCCAGGAGATTTCATAGACTACGAG

GAGTTGCGCGAGCAGCTTTCGTCTGTGAGCAGCTTCGAAAGGTTCGAGATATTCCCGAAG

GAGAGCAGCTGGCCGAATCATAACACTAACGGTGTGACAGCCGCCTGCAGTCATGAAGGA

AAGAGTTCATTCTATCGCAACCTGCTGTGGTTGACGGAGAAAGAGGGCAGCTACCCTAAG

TTGAAGAACTCCTATGTGAACAAAAAAGGCAAGGAGGTTCTGGTGCTGTGGGGCATACAC

CACCCCCCCAATAGCAAGGAGCAGCAGAATCTGTACCAAAACGAGAATGCCTATGTGAGC

GTGGTCACTAGTAACTATAACCGTCGGTTCACTCCCGAGATCGCCGAGCGTCCGAAGGTG

AGGGACCAGGCAGGCCGGATGAACTACTACTGGACCCTATTGAAGCCAGGGGACACGATT

ATCTTCGAGGCAAACGGAAACCTCATAGCGCCGATGTACGCCTTCGCCCTGAGCCGTGGC

TTTGGATCGGGGATCATCACGTCTAACGCCTCGATGCACGAATGTAATACCAAATGCCAG

ACCCCACTGGGTGCTATCAACTCGTCCTTACCCTATCAAAATATACATCCGGTCACCATA

GGCGAGTGTCCCAAATATGTCAGATCCGCCAAGTTGCGGATGGTGACCGGCCTCCGCAAT

ATTCCTAGTATTCAGTCACGCGGCTTGTTCGGCGCCATCGCTGGTTTCATCGAAGGCGGG

TGGACAGGCATGATTGATGGCTGGTATGGCTATCACCACCAGAACGAGCAGGGCTCGGGC

TACGCGGCTGACCAGAAGTCGACTCAGAATGCCATCAATGGCATCACGAATAAGGTGAAC

ACGGTCATTGAAAAGATGAACATTCAATTTACAGCCGTAGGAAAAGAGTTTAATAAACTG

GAAAAAAGAATGGAGAATCTGAATAAGAAGGTGGACGACGGATTTTTGGACATCTGGACG

TACAACGCCGAGCTGCTGGTTCTGCTGGAAAATGAGCGAACACTGGATTTTCATGATTCT

AACGTAAAGAATCTGTACGAGAAGGTGAAGTCCCAACTAAAGAATAATGCCAAGGAAATC

GGAAATGGATGCTTTGAGTTTTACCACAAGTGCGATAATGAGTGCATGGAATCCGTGCGA

AATGGTACATACGATTACCCAAAGTACTCCGAAGAATCCAAGCTAAATCGCGAAAAGGTT

GATGGTGTTAAACTTGAATCCATGGGTATTTACCAACACCATCATCACCACCATTAATAG

SEQ ID NO:137 HA1-1PR8 (no his label)

ATGAAATTCTTAGTCAACGTTGCCCTTGTTTTTATGGTCGTATACATTTCTTACATCTAT

GCGAGCCACAACGGAAAGCTGTGCCGTCTGAAAGGCATAGCGCCACTGCAGCTCGGCAAA

TGTAACATCGCAGGTTGGCTCCTTGGTAACCCGGAGTGCGACCCCCTCCTCCCTGTACGA

TCCTGGAGTTATATCGTGGAGACCCCCAATAGCGAGAACGGAATTTGCTACCCAGGAGAT

TTCATAGACTACGAGGAGTTGCGCGAGCAGCTTTCGTCTGTGAGCAGCTTCGAAAGGTTC

GAGATATTCCCGAAGGAGAGCAGCTGGCCGAATCATAACACTAACGGTGTGACAGCCGCC

TGCAGTCATGAAGGAAAGAGTTCATTCTATCGCAACCTGCTGTGGTTGACGGAGAAAGAG

GGCAGCTACCCTAAGTTGAAGAACTCCTATGTGAACAAAAAAGGCAAGGAGGTTCTGGTG

CTGTGGGGCATACACCACCCCCCCAATAGCAAGGAGCAGCAGAATCTGTACCAAAACGAG

AATGCCTATGTGAGCGTGGTCACTAGTAACTATAACCGTCGGTTCACTCCCGAGATCGCC

GAGCGTCCGAAGGTGAGGGACCAGGCAGGCCGGATGAACTACTACTGGACCCTATTGAAG

CCAGGGGACACGATTATCTTCGAGGCAAACGGAAACCTCATAGCGCCGATGTACGCCTTC

GCCCTGAGCCGTGGCTTTGGATCGGGGATCATCACGTCTAACGCCTCGATGCACGAATGT

AATACCAAATGCCAGACCCCACTGGGTGCTATCAACTCGTCCTTACCCTATCAAAATATA

CATCCGGTCACCATAGGCGAGTGTCCCAAATATGTCAGATAATAG

SEQ ID NO:138 REV primer

ACCTGCATGCCTATTATCTGACATATTTGGGACACTCGCCTATGG

SEQ ID NO：139 HA1-1NC

ATGAAATTCTTAGTCAACGTTGCCCTTGTTTTTATGGTCGTATACATTTCTTACATCTAT

GCGAGCCACAATGGTAAGCTGTGTCTGTTGAAGGGAATTGCACCCCTCCAACTGGGCAAC

TGTTCGGTCGCTGGTTGGATCCTCGGAAACCCAGAATGCGAGCTCCTTATCAGTAAGGAA

TCTTGGTCTTATATTGTCGAAACCCCGAACCCCGAGAACGGAACATGCTACCCGGGTTAC

TTTGCTGATTACGAAGAGCTTCGCGAGCAACTCAGCTCCGTATCCTCCTTCGAGCGCTTC

GAGATTTTTCCCAAAGAGTCCAGCTGGCCAAATCATACCGTCACCGGCGTGTCGGCCTCC

TGTTCCCACAACGGAAAGTCTAGCTTCTATAGAAATCTTCTCTGGCTGACGGGTAAGAAT

GGTCTTTACCCCAATTTGAGCAAGTCCTACGTCAACAACAAAGAAAAGGAAGTTCTGGTA

TTGTGGGGTGTGCACCACCCTCCGAACATCGGCAATCAGCGCGCCCTGTATCACACAGAG

AACGCGTATGTTTCCGTTGTCTCCTCACATTACTCGAGGCGCTTCACTCCTGAAATAGCT

AAGCGTCCGAAAGTGCGTGACCAGGAGGGACGTATCAACTATTATTGGACGCTGTTGGAG

CCAGGCGATACAATTATCTTCGAGGCTAACGGTAACCTTATCGCTCCCTGGTACGCCTTC

GCCCTGTCGCGTGGTTTCGGTAGTGGAATAATCACTAGTAATGCTCCTATGGACGAGTGT

GACGCTAAGTGCCAAACACCTCAGGGCGCTATCAATAGCTCCCTTCCATTCCAGAACGTC

CATCCGGTTACCATTGGAGAGTGTCCAAAGTACGTGAGACACCATCATCACCATCACTAA

TAG

SEQ ID NO:140 FOR primer

AGGCAGATCTATGAAATTCTTAGTCAACGTTGCCCTTGTTTTTATGGTCGTATACATTTC

TTACATCTATGCGAGCCACAATGGTAAGCTGTGTCTGTTGAAG

SEQ ID NO:141 REV primer

AGTCGCATGCCTATTAATGGTGGTGATGATGGTGTCTCACGTACTTTGGACACTCTCCAATGG

SEQ ID NO：142 HA0s NC

AGATCTATGAAGTTTCTCGTGAACGTTGCACTGGTGTTTATGGTAGTCTATATATCGTAC

ATTTATGCTGACACGATTTGCATCGGTTACCATGCGAACAACTCCACGGATACCGTGGAC

ACAGTGTTGGAAAAGAACGTGACCGTCACGCACTCCGTAAACCTTCTGGAGGACAGCCAC

AATGGTAAGCTGTGTCTGTTGAAGGGAATTGCACCCCTCCAACTGGGCAACTGTTCGGTC

GCTGGTTGGATCCTCGGAAACCCAGAATGCGAGCTCCTTATCAGTAAGGAATCTTGGTCT

TATATTGTCGAAACCCCGAACCCCGAGAACGGAACATGCTACCCGGGTTACTTTGCTGAT

TACGAAGAGCTTCGCGAGCAACTCAGCTCCGTATCCTCCTTCGAGCGCTTCGAGATTTTT

CCCAAAGAGTCCAGCTGGCCAAATCATACCGTCACCGGCGTGTCGGCCTCCTGTTCCCAC

AACGGAAAGTCTAGCTTCTATAGAAATCTTCTCTGGCTGACGGGTAAGAATGGTCTTTAC

CCCAATTTGAGCAAGTCCTACGTCAACAACAAAGAAAAGGAAGTTCTGGTATTGTGGGGT

GTGCACCACCCTCCGAACATCGGCAATCAGCGCGCCCTGTATCACACAGAGAACGCGTAT

GTTTCCGTTGTCTCCTCACATTACTCGAGGCGCTTCACTCCTGAAATAGCTAAGCGTCCG

AAAGTGCGTGACCAGGAGGGACGTATCAACTATTATTGGACGCTGTTGGAGCCAGGCGAT

ACAATTATCTTCGAGGCTAACGGTAACCTTATCGCTCCCTGGTACGCCTTCGCCCTGTCG

CGTGGTTTCGGTAGTGGAATAATCACTAGTAATGCTCCTATGGACGAGTGTGACGCTAAG

TGCCAAACACCTCAGGGCGCTATCAATAGCTCCCTTCCATTCCAGAACGTCCATCCGGTT

ACCATTGGAGAGTGTCCAAAGTACGTGAGATCGGCCAAACTTCGCATGGTCACGGGTCTG

CGCAACATCCCGTCAATCCAATCTAGGGGCCTCTTCGGCGCTATCGCCGGTTTCATTGAG

GGCGGTTGGACTGGAATGGTTGACGGATGGTACGGCTATCATCACCAGAACGAACAAGGT

TCCGGTTACGCTGCTGACCAGAAATCTACTCAGAACGCGATCAATGGTATCACGAACAAG

GTGAACAGCGTCATTGAAAAGATGAATACTCAGTTTACAGCCGTGGGCAAAGAGTTCAAT

AAACTCGAGAGACGTATGGAAAACCTCAATAAGAAGGTGGATGACGGCTTCCTGGACATT

TGGACTTACAACGCCGAGCTGCTGGTCCTGCTCGAGAACGAGAGAACCCTTGACTTCCAC

GACAGCAACGTCAAGAACCTGTACGAGAAGGTGAAAAGTCAACTTAAAAACAATGCCAAG

GAGATTGGTAACGGCTGCTTCGAATTCTACCACAAGTGTAATAATGAGTGCATGGAATCC

GTTAAGAACGGCACCTACGATTACCCTAAATACTCAGAGGAGTCCAAGCTTAACCGCGAG

AAGATCGACGGCGTAAAACTGGAAAGCATGGGCGTATACCAACACCATCATCACCATCAC

TAATAGGCATGC

SEQ ID NO：143 HA1-1VN

ATGAAATTCTTAGTCAACGTTGCCCTTGTTTTTATGGTCGTATACATTTCTTACATCTAT

GCGGAGAAGAAACACAACGGTAAGCTTTGCGACTTGGATGGAGTCAAGCCCCTCATACTT

AGAGATTGTAGTGTAGCCGGTTGGCTGCTCGGTAACCCAATGTGCGATGAGTTCATCAAT

GTTCCCGAATGGTCATATTATCGTCGAAAAAGCTAATCCTGTCAACGACCTGTGCTACCCC

GGTGATTTCAATGACTATGAAGAACTGAAGCACCTGCTCTCCCGCATCAACCATTTCGAG

AAAATCCAGATCATTCCCAAGAGTTCCTGGTCTAGCCATGAGGCTAGTCTGGGTGTCTCA

TCCGCCTGCCCATATCAGGGTAAAAGTTCTTTCTTTAGGAACGTAGTATGGTTGATAAAG

AAAAACTCTACATACCCGACCATCAAGCGCTCTTACAACAATACGAACCAAGAGGATCTG

CTTGTCCTTTGGGGAATCCATCATCCTAATGATGCTGCCGAACAGACTAAGCTCTACCAA

AACCCTACCACTTATATTTCCGTCGGCACCTCTACTCTGAACCAGCGCCTTGTGCCCAGG

ATCGCTACGAGATCAAAAGTCAACGGCCAATCGGGCCGCATGGAATTCTTCTGGACGATC

CTGAAGCCTAATGACGCTATCAACTTCGAGTCAAATGGAAACTTTATCGCTCCCGAGTAC

GCTTACAAGATCGTCAAGAAGGGCGACTCCACGATTATGAAGTCAGAGTTGGAGTACGGC

AACTGCAACACAAAGTGCCAAACTCCTATGGGCGCTATAAATTCTTCAATGCCGTTCCAC

AACATCCATCCGCTCACGATCGGTGAGTGCCCGAAATATGTAAAGCACCATCACCACCAT

CACTAATAG

SEQ ID NO:144 FOR primer

AGGCAGATCTATGAAATTCTTAGTCAACGTTGCCCTTGTTTTTATGGTCGTATACATTTC

TTACATCTATGCGGAGAAGAAACACAACGGTAAGCTTTG

SEQ ID NO:145 REV primer

AGTCGCATGCCTATTATGGTGGTGATGATGGTGCTTTACATATTTCGGGCACTCACCG

SEQ ID NO：146 HA0s VN

AGATCTATGAAATTCTTGGTTAATGTAGCCCTGGTGTTTATGGTAGTGTACATTTCATAC

ATTTATGCTGATCAAATCTGCATTGGCTACCATGCCAACAACAGCACCGAGCAAGTTGAC

ACGATCATGGAGAAGAACGTAACCGTCACTCACGCTCAAGACATCCTGGAGAAGAAACAC

AACGGTAAGCTTTGCGACTTGGATGGAGTCAAGCCCCTCATACTTAGAGATTGTAGTGTA

GCCGGTTGGCTGCTCGGTAACCCAATGTGCGATGAGTTCATCAATGTTCCCGAATGGTCA

TATATCGTCGAAAAAGCTAATCCTGTCAACGACCTGTGCTACCCCGGTGATTTCAATGAC

TATGAAGAACTGAAGCACCTGCTCTCCCGCATCAACCATTTCGAGAAAATCCAGATCATT

CCCAAGAGTTCCTGGTCTAGCCATGAGGCTAGTCTGGGTGTCTCATCCGCCTGCCCATAT

CAGGGTAAAAGTTCTTTCTTTAGGAACGTAGTATGGTTGATAAAGAAAAACTCTACATAC

CCGACCATCAAGCGCTCTTACAACAATACGAACCAAGAGGATCTGCTTGTCCTTTGGGGA

ATCCATCATCCTAATGATGCTGCCGAACAGACTAAGCTCTACCAAAACCCTACCACTTAT

ATTTCCGTCGGCACCTCTACTCTGAACCAGCGCCTTGTGCCCAGGATCGCTACGAGATCA

AAAGTCAACGGCCAATCGGGCCGCATGGAATTCTTCTGGACGATCCTGAAGCCTAATGAC

GCTATCAACTTCGAGTCAAATGGAAACTTTATCGCTCCCGAGTACGCTTACAAGATCGTC

AAGAAGGGCGACTCCACGATTATGAAGTCAGAGTTGGAGTACGGCAACTGCAACACAAAG

TGCCAAACTCCTATGGGCGCTATAAATTCTTCAATGCCGTTCCACAACATCCATCCGCTC

ACGATCGGTGAGTGCCCGAAATATGTAAAGTCGAATCGTCTCGTACTGGCGACAGGCCTG

AGAAATAGTCCGCAACGTGAACGTCGTCGCAAGAAGAGAGGACTGTTTGGTGCCATTGCA

GGCTTTATTGAGGGCGGCTGGCAGGGCATGGTTGACGGATGGTACGGCTACCACCATTCA

AACGAGCAGGGATCTGGCTACGCCGCTGACAAAGAAAGCACCCAAAAGGCCATTGATGGA

GTGACGAATAAGGTGAATTCGATCATCGACAAAATGAACACGCAATTCGAAGCAGTGGGT

CGCGAATTCAATAACCTGGAGCGCCGTATCGAGAATCTGAACAAGAAGATGGAAGACGGC

TTTTTGGATGTCTGGACATATAACGCTGAATTGCTGGTCCTCATGGAAAACGAGCGATCC

CTTGATTTCCACGACAGCAACGTTAAGAACCTCTACGACAAGGTCAGGCTCCAGCTCAGG

GATAACGCCAAGGAATTGGGAAACGGATGCTTCGAGTTCTACCACAAATGCGACAACGAG

TGCATGGAGTCAGTCAGGAATGGTACCTACGACTACCCGCAATATTCTGAGGAGGCTCGC

TTGAAGCGTGAGGAAATATCGGGTGTTAAATTGGAGAGTATTGGAATCTACCAGCACCAT

CACCACCATCACTAATAGGCATGC

SEQ ID NO：147 HA1-1 IND

ATGAAATTCTTAGTCAACGTTGCCCTTGTTTTTATGGTCGTATACATTTCTTACATCTAT

GCGGAGAAAACCCATAACGGTAAGTTGTGCGACCTTGACGGTGTAAAGCCCCTGATCCTC

CGTGACTGCAGTGTTGCTGGTTGGCTTTTGGGCAACCCCATGTGTGACGAATTTATCAAC

GTGCCTGAATGGTCATACATTGTAGAGAAGGCCAACCCCACGAACGATCTCTGTTATCCC

GGCAGCTTCAATGACTATGAGGAACTTAAGCACCTTCTGTCACGTATCAACCACTTCGAA

AAGATCCAGATCATCCCGAAGAGCTCCTGGAGCGACCACGAAGCCAGTTCGGGTGTGTCT

TCCGCTTGCCCCTACCTCGGTAGCCCTTCCTTCTTCCGTAACGTAGTGTGGCTGATCAAG

AAGAATAGCACTTACCCTACAATCAAAAAGTCGTATAACAATACTAACCAAGAGGATCTG

CTTGTACTCTGGGGAATTCATCATCCCAACGACGCGGCGGAGCAGACCAGGTTGTACCAG

AACCCCACCACTTACATCTCCATCGGTACGTCCACACTGAATCAGCGTCTGGTCCCCAAG

ATCGCAACCAGGTCCAAGGTTAACGGTCAGTCCGGTCGTATGGAGTTCTTCTGGACCATC

CTGAAGCCCAACGACGCCATCAACTTCGAGTCCAACGGTAATTTCATTGCTCCGGAGTAC

GCCTACAAGATAGTTAAGAAGGGTGATTCAGCGATCATGAAGTCGGAACTTGAGTATGGC

AACTGCAACACTAAATGCCAAACTCCAATGGGCGCTATCAACTCCAGTATGCCATTCCAT

AACATCCACCCATTGACAATCGGTGAATGTCCCAAGTACGTGAAGCACCACCATCACCAT

CACTAATAG

SEQ ID NO:148 FOR primer

AGGCAGATCTATGAAATTCTTAGTCAACGTTGCCCTTGTTTTTATGGTCGTATACATTTC

TTACATCTATGCGGAGAAAACCCATAACGGTAAGTTGTG

SEQ ID NO:149 REV primer

AGTCGCATGCCTATTAATGGTGGTGATGATGGTGCTTCACGTACTTGGGACATTCACCGA

TTG

SEQ ID NO：150 HA0s IND

AGATCTATGAAGTTCCTGGTCAATGTAGCCTTGGTATTTATGGTAGTCTATATCTCGTAC

ATTTACGCAGACCAGATTTGTATTGGATATCACGCTAACAACAGCACAGAGCAGGTAGAT

ACTATTATGGAGAAAAATGTTACCGTCACTCACGCCCAGGACATCCTGGAGAAAACCCAT

AACGGTAAGTTGTGCGACCTTGACGGTGTAAAGCCCCTGATCCTCCGTGACTGCAGTGTT

GCTGGTTGGCTTTTGGGCAACCCCATGTGTGACGAATTTATCAACGTGCCTGAATGGTCA

TACATTGTAGAGAAGGCCAACCCCACGAACGATCTCTGTTATCCCGGCAGCTTCAATGAC

TATGAGGAACTTAAGCACCTTCTGTCACGTATCAACCACTTCGAAAAGATCCAGATCATC

CCGAAGAGCTCCTGGAGCGACCACGAAGCCAGTTCGGGTGTGTCTTCCGCTTGCCCCTAC

CTCGGTAGCCCTTCCTTCTTCCGTAACGTAGTGTGGCTGATCAAGAAGAATAGCACTTAC

CCTACAATCAAAAAGTCGTATAACAATACTAACCAAGAGGATCTGCTTGTACTCTGGGGA

ATTCATCATCCCAACGACGCGGCGGAGCAGACCAGGTTGTACCAGAACCCCACCACTTAC

ATCTCCATCGGTACGTCCACACTGAATCAGCGTCTGGTCCCCAAGATCGCAACCAGGTCC

AAGGTTAACGGTCAGTCCGGTCGTATGGAGTTCTTCTGGACCATCCTGAAGCCCAACGAC

GCCATCAACTTCGAGTCCAACGGTAATTTCATTGCTCCGGAGTACGCCTACAAGATAGTT

AAGAAGGGTGATTCAGCGATCATGAAGTCGGAACTTGAGTATGGCAACTGCAACACTAAA

TGCCAAACTCCAATGGGCGCTATCAACTCCAGTATGCCATTCCATAACATCCACCCATTG

ACAATCGGTGAATGTCCCAAGTACGTGAAGAGCAACAGGTTGGTATTGGCCACCGGTTTG

AGAAACAGCCCCCAGAGAGAGTCGCGTCGTAAAAAGCGCGGCTTGTTCGGAGCCATCGCT

GGCTTCATAGAGGGTGGTTGGCAGGGAATGGTCGATGGTTGGTATGGTTATCATCATTCC

AACGAGCAGGGAAGTGGTTACGCCGCCGACAAAGAATCGACCCAGAAGGCTATTGACGGC

GTCACAAACAAAGTAAACTCTATCATTGATAAGATGAACACCCAGTTCGAGGCTGTAGGT

AGAGAATTCAACAACCTCGAAAGACGTATTGAGAACCTGAACAAGAAAATGGAGGATGGC

TTCCTGGACGTGTGGACCTACAATGCTGAGCTGTTGGTCCTTATGGAGAACGAGCGTACC

CTCGATTTCCATGACTCAAACGTGAAGAACCTGTATGACAAGGTGCGTTTGCAACTGAGG

GACAACGCAAAGGAGCTTGGAAACGGTTGTTTCGAATTTTATCATAAGTGCGACAATGAG

TGTATGGAGTCGATTAGAAATGGCACGTACAACTACCCTCAATACAGCGAAGAAGCTCGT

CTCAAACGTGAGGAAATCAGCGGCGTCAAGCTCGAATCAATCGGTACCTATCAGCACCAC

CATCACCATCACTAATAGGCATGC

SEQ ID NO：151 STF2.HA1-1His(PR8)

MKFLVNVALVFMVVYISYIYAAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAA

GQAIANRFTANIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQ

AEITQRLNEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDSLNVQKAY

DVKDTAVTTKAYANNGTTLDVSGLDDAAIKAATGGTNGTASVTGGAVKFDADNNKYFVTIGGFT

GADAAKNGDYEVNVATDGTVTLAAGATKTTMPAGATTKTEVQELKDTPAVVSADAKNALIAGG

VDATDANGAELVKMSYTDKNGKTIEGGYALKAGDKYYAADYDEATGAIKAKTTSYTAADGTTK

TAANQLGGVDGKTEVVTIDGKTYNASKAAGHDFKAQPELAEAAAKTTENPLQKIDAALAQVDA

LRSDLGAVQNRFNSAITNLGNTVNNLSEARSRIEDSDYATEVSNMSRAQILQQAGTSVLAQANQV

PQNVLSLLASGSGSGSSHNGKLCRLKGIAPLQLGKCNIAGWLLGNPECDPLLPVRSWSYIVETPNS

ENGICYPGDFIDYEELREQLSSVSSFERFEIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTE

KEGSYPKLKNSYVNKKGKEVLVLWGIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAERP

KVRDQAGRMNYYWTLLKPGDTIIFEANGNLIAPMYAFALSRGFGSGIITSNASMHECNTKCQTPL

GAINSSLPYQNIHPVTIGECPKYVRHHHHHH

SEQ ID NO：152 ngSTF2.HA1-1His(PR8)

MKFLVNVALVFMVVYISYIYAAQVINTNSLSLLTQNNLQKSQSALGTAIERLSSGLRINSAKDDAA

GQAIANRFTANIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSAQSTNSQSDLDSIQ

AEITQRLNEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDSLNVQKAY

DVKDTAVTTKAYANQGTTLDVSGLDDAAIKAATGGTQGTASVTGGAVKFDADNNKYFVTIGGFT

GADAAKNGDYEVNVATDGTVTLAAGATKTTMPAGATTKTEVQELKDTPAVVSADAKNALIAGG

VDATDANGAELVKMSYTDKNGKTIEGGYALKAGDKYYAADYDEATGAIKAKTTSYTAADGTTK

TAANQLGGVDGKTEVVTIDGKTYQASKAAGHDFKAQPELAEAAAKTTENPLQKIDAALAQVDA

LRSDLGAVQNRFNSAITNLGNTVNQLSEARSRIEDSDYATEVSQMSRAQILQQAGTSVLAQANQV

PQNVLSLLASGSGSGSSHNGKLCRLKGIAPLQLGKCNIAGWLLGNPECDPLLPVRSWSYIVETPNS

ENGICYPGDFIDYEELREQLSSVSSFERFEIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTE

KEGSYPKLKNSYVNKKGKEVLVLWGIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAERP

KVRDQAGRMNYYWTLLKPGDTIIFEANGNLIAPMYAFALSRGFGSGIITSQASMHECNTKCQTPL

GAIQSSLPYQNIHPVTIGECPKYVRHHHHHH

SEQ ID NO：153 STF2.HA1-2His(PR8)

MKFLVNVALVFMVVYISYIYAAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAA

GQAIANRFTANIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQ

AEITQRLNEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDSLNVQKAY

DVKDTAVTTKAYANNGTTLDVSGLDDAAIKAATGGTNGTASVTGGAVKFDADNNKYFVTIGGFT

GADAAKNGDYEVNVATDGTVTLAAGATKTTMPAGATTKTEVQELKDTPAVVSADAKNALIAGG

VDATDANGAELVKMSYTDKNGKTIEGGYALKAGDKYYAADYDEATGAIKAKTTSYTAADGTTK

TAANQLGGVDGKTEVVTIDGKTYNASKAAGHDFKAQPELAEAAAKTTENPLQKIDAALAQVDA

LRSDLGAVQNRFNSAITNLGNTVNNLSEARSRIEDSDYATEVSNMSRAQILQQAGTSVLAQANQV

PQNVLSLLASGSGSGSKGIAPQLGKCNIAGWLLGNPECDPLLPVRSWSYIVETPNSENGICYPGDFI

DYEELREQLSSVSSFERFEIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTEKEGSYPKLKN

SYVNKKGKEVLVLWGIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQAGRM

NYYWTLLKPGDTIIFEANGNLIAPMYAFALSRGFGSGIITSHHHHHH

SEQ ID NO：154 ngSTF2.HA1-2His(PR8)

MKFLVNVALVFMVVYISYIYAAQVINTNSLSLLTQNNLQKSQSALGTAIERLSSGLRINSAKDDAA

GQAIANRFTANIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSAQSTNSQSDLDSIQ

AEITQRLNEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDSLNVQKAY

DVKDTAVTTKAYANQGTTLDVSGLDDAAIKAATGGTQGTASVTGGAVKFDADNNKYFVTIGGFT

GADAAKNGDYEVNVATDGTVTLAAGATKTTMPAGATTKTEVQELKDTPAVVSADAKNALIAGG

VDATDANGAELVKMSYTDKNGKTIEGGYALKAGDKYYAADYDEATGAIKAKTTSYTAADGTTK

TAANQLGGVDGKTEVVTIDGKTYQASKAAGHDFKAQPELAEAAAKTTENPLQKIDAALAQVDA

LRSDLGAVQNRFNSAITNLGNTVNQLSEARSRIEDSDYATEVSQMSRAQILQQAGTSVLAQANQV

PQNVLSLLASGSGSGSKGIAPLQLGKCNIAGWLLGNPECDPLLPVRSWSYIVETPNSENGICYPGD

FIDYEELREQLSSVSSFERFEIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTEKEGSYPKLK

NSYVNKKGKEVLVLWGIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQAGR

MNYYWTLLKPGDTIIFEANGNLIAPMYAFALSRGFGSGIITSHHHHHH

SEQ ID NO：155 STF2.HA1-2mutHis(PR8)

MKFLVNVALVFMVVYISYIYAAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAA

GQAIANRFTANIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQ

AEITQRLNEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDSLNVQKAY

DVKDTAVTTKAYANNGTTLDVSGLDDAAIKAATGGTNGTASVTGGAVKFDADNNKYFVTIGGFT

GADAAKNGDYEVNVATDGTVTLAAGATKTTMPAGATTKTEVQELKDTPAVVSADAKNALIAGG

VDATDANGAELVKMSYTDKNGKTIEGGYALKAGDKYYAADYDEATGAIKAKTTSYTAADGTTK

TAANQLGGVDGKTEVVTIDGKTYNASKAAGHDFKAQPELAEAAAKTTENPLQKIDAALAQVDA

LRSDLGAVQNRFNSAITNLGNTVNNLSEARSRIEDSDYATEVSNMSRAQILQQAGTSVLAQANQV

PQNVLSLLASGSGSGSKGAAPLQLGKCNIAGWLLGNPECDPLLPVRSWSDIAETPNSENGICYPGD

FIDYEELREQLSSVSSFERFEIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTEKEGSYPKLK

NSYVNKKGKEVLVLWGIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQAGR

MNYYWTLLKPGDTIIFEANGNLIAPMYAFALSRGFGSGIITSHHHHHH

SEQ ID NO：156 ngSTF2.HA1-2mutHis(PR8)

MKFLVNVALVFMVVYISYIYAAQVINTNSLSLLTQNNLQKSQSALGTAIERLSSGLRINSAKDDAA

GQAIANRFTANIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSAQSTNSQSDLDSIQ

AEITQRLNEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDSLNVQKAY

DVKDTAVTTKAYANQGTTLDVSGLDDAAIKAATGGTQGTASVTGGAVKFDADNNKYFVTIGGFT

GADAAKNGDYEVNVATDGTVTLAAGATKTTMPAGATTKTEVQELKDTPAVVSADAKNALIAGG

VDATDANGAELVKMSYTDKNGKTIEGGYALKAGDKYYAADYDEATGAIKAKTTSYTAADGTTK

TAANQLGGVDGKTEVVTIDGKTYQASKAAGHDFKAQPELAEAAAKTTENPLQKIDAALAQVDA

LRSDLGAVQNRFNSAITNLGNTVNQLSEARSRIEDSDYATEVSQMSRAQILQQAGTSVLAQANQV

PQNVLSLLASGSGSGSKGAAPQLGKCNIAGWLLGNPECDPLLPVRSWSDIAETPNSENGICYPGDF

IDYEELREQLSSVSSFERFEIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTEKEGSYPKLKN

SYVNKKGKEVLVLWGIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQAGRM

NYYWTLLKPGDTIIFEANGNLIAPMYAFALSRGFGSGIITSHHHHHH

SEQ ID NO：157 STF2.HA1-3His(PR8)

MKFLVNVALVFMVVYIS YIYAAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAA

GQAIANRFTANIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQ

AEITQRLNEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDSLNVQKAY

DVKDTAVTTKAYANNGTTLDVSGLDDAAIKAATGGTNGTASVTGGAVKFDADNNKYFVTIGGFT

GADAAKNGDYEVNVATDGTVTLAAGATKTTMPAGATTKTEVQELKDTPAVVSADAKNALIAGG

VDATDANGAELVKMSYTDKNGKTIEGGYALKAGDKYYAADYDEATGAIKAKTTSYTAADGTTK

TAANQLGGVDGKTEVVTIDGKTYNASKAAGHDFKAQPELAEAAAKTTENPLQKIDAALAQVDA

LRSDLGAVQNRFNSAITNLGNTVNNLSEARSRIEDSDYATEVSNMSRAQILQQAGTSVLAQANQV

PQNVLSLLASGSGSGSNSENGCYPGDFIDYEELREQLSSVSSFERFEIFPKESSWPNHNTNGVTAAC

SHEGKSSFYRNLLWLTEKEGSYPKLKNSYVNKKGKEVLVLWGIHHPPNSKEQQNLYQNENAYVS

VVTSNYNRRFTPEIAERPKVRDQAGRMNYYWTLLKPGDTIIFEANGNLIAPMYAFALSRGHHHHH

H

SEQ ID NO：158 STF2.HA1-3mutHis(PR8)

MKFLVNVALVFMVVYISYIYAAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAA

GQAIANRFTANIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQ

AEITQRLNEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDSLNVQKAY

DVKDTAVTTKAYANNGTTLDVSGLDDAAIKAATGGTNGTASVTGGAVKFDADNNKYFVTIGGFT

GADAAKNGDYEVNVATDGTVTLAAGATKTTMPAGATTKTEVQELKDTPAVVSADAKNALIAGG

VDATDANGAELVKMSYTDKNGKTIEGGYALKAGDKYYAADYDEATGAIKAKTTSYTAADGTTK

TAANQLGGVDGKTEVVTIDGKTYNASKAAGHDFKAQPELAEAAAKTTENPLQKIDAALAQVDA

LRSDLGAVQNRFNSAITNLGNTVNNLSEARSRIEDSDYATEVSNMSRAQILQQAGTSVLAQANQV

PQNVLSLLASGSGSGSNSENEICYPGDFIDKEELREQLSSVSSFERFEIFPKESSWPNHNTNGVTAA

CSHEGKSSFYRNLLWLTEKEGSYPKLKNSYVNKKGKEVLVLWGIHHPPNSKEQQNLYQNENAYV

SVVTSNYNRRFTPEIAERPKVRDQAGRMNYYWTLLKPGDTIIFEANGNLIAPMYAAALSRGHHHH

HH

SEQ ID NO：159 STF2.HA1-2(IND)

MAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANRFTANIKGLTQASR

NANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLNEIDRVSGQTQFN

GVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDSLNVQKAYDVKDTAVTTKAYANNGTTL

DVSGLDDAAIKAATGGTNGTASVTGGAVKFDADNNKYFVTIGGFTGADAAKNGDYEVNVATDG

TVTLAAGATKTTMPAGATTKTEVQELKDTPAVVSADAKNALIAGGVDATDANGAELVKMSYTDK

NGKTIEGGYALKAGDKYYAADYDEATGAIKAKTTSYTAADGTTKTAANQLGGVDGKTEVVTIDG

KTYNASKAAGHDFKAQPELAEAAAKTTENPLQKIDAALAQVDALRSDLGAVQNRFNSAITNLGN

TVNNLSEARSRIEDSDYATEVSNMSRAQILQQAGTSVLAQANQVPQNVLSLLAGVKPLILRDCSV

AGWLLGNPMCDEFINVPEWSYIVEKANPTNDLCYPGSFNDYEELKHLLSRINHFEKIQIIPKSSWS

DHEASSGVSSACPYLGSPSFFRNVVWLIKKNSTYPTIKKSYNNTNQEDLLVLWGIHHPNDAAEQT

RLYQNPTTYISIGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKI

VKKGDSAIMKSE

Drosophila

SEQ ID NO：160 STF2Δ.HA0shis

ATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAA

TCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGC

GCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGT

CTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGC

GCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCT

AACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTG

AACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAG

GACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTG

AAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCATGGAGCGCCGGTG

GATCCTGCTAGCCCATGGACCGAAAACCCGCTGCAGAAAATTGATGCCGCGCTGGCGCAG

GTGGATGCGCTGCGCTCTGATCTGGGTGCGGTACAAAACCGTTTCAACTCTGCTATCACC

AACCTGGGCAATACCGTAAACAATCTGTCTGAAGCGCGTAGCCGTATCGAAGATTCCGAC

TACGCGACCGAAGTTTCCAACATGTCTCGCGCGCAGATTTTGCAGCAGGCCGGTACTTCC

GTTCTGGCGCAGGCTAACCAGGTCCCGCAGAACGTGCTGTCTCTGTTACGTGAATTCTCT

AGATATCCAGCACAGTGGCGGCCGCTCGACACAATATGTATAGGCTACCATGCGAACAAT

TCAACCGACACTGTTGACACAGTACTCGAGAAGAATGTGACAGTGACACACTCTGTTAAC

CTGCTCGAAGACAGCCACAACGGAAAACTATGTAGATTAAAAGGAATAGCCCCACTACAA

TTGGGGAAATGTAACATCGCCGGATGGCTTTTGGGAAACCCAGAATGCGACCCACTGCTT

CCAGTGAGATCATGGTCCTACATTGTAGAAACACCAAACTCTGAGAATGGAATATGTTAT

CCAGGAGATTTCATCGACTATGAGGAGCTGAGGGAGCAATTGAGCTCAGTGTCATCATTC

GAAAGATTCGAAATATTTCCCAAAGAAAGCTCATGGCCCAACCACAACACAAACGGAGTA

ACGGCAGCATGCTCCCATGAGGGGAAAAGCAGTTTTTACAGAAATTTGCTATGGCTGACG

GAGAAGGAGGGCTCATACCCAAAGCTGAAAAATTCTTATGTGAACAAAAAAGGGAAAGAA

GTCCTTGTACTGTGGGGTATTCATCACCCGCCTAACAGTAAGGAACAACAGAATCTCTAT

CAGAATGAAAATGCTTATGTCTCTGTAGTGACTTCAAATTATAACAGGAGATTTACCCCG

GAAATAGCAGAAAGACCCAAAGTAAGAGATCAAGCTGGGAGGATGAACTATTACTGGACC

TTGCTAAAACCCGGAGACACAATAATATTTGAGGCAAATGGAAATCTAATAGCACCAATG

TATGCTTTCGCACTGAGTAGAGGCTTTGGGTCCGGCATCATCACCTCAAACGCATCAATG

CATGAGTGTAACACGAAGTGTCAAACACCCCTGGGAGCTATAAACAGCAGTCTCCCTTAC

CAGAATATACACCCAGTCACAATAGGAGAGTGCCCAAAATACGTCAGGAGTGCCAAATTG

AGGATGGTTACAGGACTAAGGAACATTCCGTCCATTCAATCCAGAGGTCTATTTGGAGCC

ATTGCCGGTTTTATTGAAGGGGGATGGACTGGAATGATAGATGGATGGTATGGTTATCAT

CATCAGAATGAACAGGGATCAGGCTATGCAGCGGATCAAAAAAGCACACAAAATGCCATT

AACGGGATTACAAACAAGGTGAACACTGTTATCGAGAAAATGAACATTCAATTCACAGCT

GTGGGTAAAGAATTCAACAAATTAGAAAAAAGGATGGAAAATTTAAATAAAAAAGTTGAT

GATGGATTTCTGGACATTTGGACATATAATGCAGAATTGTTAGTTCTACTGGAAAATGAA

AGGACTCTGGATTTCCATGACTCAAATGTGAAGAATCTGTATGAGAAAGTAAAAAGCCAA

TTAAAGAATAATGCCAAAGAAATCGGAAATGGATGTTTTGAGTTCTACCACAAGTGTGAC

AATGAATGCATGGAAAGTGTAAGAAATGGGACTTATGATTATCCCAAATATTCAGAAGAG

TCAAAGTTGAACAGGGAAAAGGTAGATGGAGTGAAATTGGAATCAATGGGGATCTATCAG

ACGCGTACCGGTCATCATCACCATCACCATTGA

SEQ ID NO:161 FOR primer

ATTCTCCGGCGGCCGCTCGACACAATATGTATAGGCTACC

SEQ ID NO:162 REV primer

AGTCTTGCGGCCGCCTATTAATGGTGATGGTGATGATGCTGATAGATCCCCATTGATTCC

SEQ ID NO:163 HA0s PR8 template

GACACAATATGTATAGGCTACCATGCGAACAATTCAACCGACACTGTTGACACAGTACTC

GAGAAGAATGTGACAGTGACACACTCTGTTAACCTGCTCGAAGACAGCCACAACGGAAAA

CTATGTAGATTAAAAGGAATAGCCCCACTACAATTGGGGAAATGTAACATCGCCGGATGG

CTTTTGGGAAACCCAGAATGCGACCCACTGCTTCCAGTGAGATCATGGTCCTACATTGTA

GAAACACCAAACTCTGAGAATGGAATATGTTATCCAGGAGATTTCATCGACTATGAGGAG

CTGAGGGAGCAATTGAGCTCAGTGTCATCATTCGAAAGATTCGAAATATTTCCCAAAGAA

AGCTCATGGCCCAACCACAACACAAACGGAGTAACGGCAGCATGCTCCCATGAGGGGAAA

AGCAGTTTTTACAGAAATTTGCTATGGCTGACGGAGAAGGAGGGCTCATACCCAAAGCTG

AAAAATTCTTATGTGAACAAAAAAGGGAAAGAAGTCCTTGTACTGTGGGGTATTCATCAC

CCGCCTAACAGTAAGGAACAACAGAATCTCTATCAGAATGAAAATGCTTATGTCTCTGTA

GTGACTTCAAATTATAACAGGAGATTTACCCCGGAAATAGCAGAAAGACCCAAAGTAAGA

GATCAAGCTGGGAGGATGAACTATTACTGGACCTTGCTAAAACCCGGAGACACAATAATA

TTTGAGGCAAATGGAAATCTAATAGCACCAATGTATGCTTTCGCACTGAGTAGAGGCTTT

GGGTCCGGCATCATCACCTCAAACGCATCAATGCATGAGTGTAACACGAAGTGTCAAACA

CCCCTGGGAGCTATAAACAGCAGTCTCCCTTACCAGAATATACACCCAGTCACAATAGGA

GAGTGCCCAAAATACGTCAGGAGTGCCAAATTGAGGATGGTTACAGGACTAAGGAACATT

CCGTCCATTCAATCCAGAGGTCTATTTGGAGCCATTGCCGGTTTTATTGAAGGGGGATGG

ACTGGAATGATAGATGGATGGTATGGTTATCATCATCAGAATGAACAGGGATCAGGCTAT

GCAGCGGATCAAAAAAGCACACAAAATGCCATTAACGGGATTACAAACAAGGTGAACACT

GTTATCGAGAAAATGAACATTCAATTCACAGCTGTGGGTAAAGAATTCAACAAATTAGAA

AAAAGGATGGAAAATTTAAATAAAAAAGTTGATGATGGATTTCTGGACATTTGGACATAT

AATGCAGAATTGTTAGTTCTACTGGAAAATGAAAGGACTCTGGATTTCCATGACTCAAAT

GTGAAGAATCTGTATGAGAAAGTAAAAAGCCAATTAAAGAATAATGCCAAAGAAATCGGA

AATGGATGTTTTGAGTTCTACCACAAGTGTGACAATGAATGCATGGAAAGTGTAAGAAAT

GGGACTTATGATTATCCCAAATATTCAGAAGAGTCAAAGTTGAACAGGGAAAAGGTAGAT

GGAGTGAAATTGGAATCAATGGGGATCTATCAG

SEQ ID NO：164 STF2Δ.HA0s

ATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAA

TCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGC

GCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGT

CTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGC

GCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCT

AACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTG

AACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAG

GACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTG

AAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCATGGAGCGCCGGTG

GATCCTGCTAGCCCATGGACCGAAAACCCGCTGCAGAAAATTGATGCCGCGCTGGCGCAG

GTGGATGCGCTGCGCTCTGATCTGGGTGCGGTACAAAACCGTTTCAACTCTGCTATCACC

AACCTGGGCAATACCGTAAACAATCTGTCTGAAGCGCGTAGCCGTATCGAAGATTCCGAC

TACGCGACCGAAGTTTCCAACATGTCTCGCGCGCAGATTTTGCAGCAGGCCGGTACTTCC

GTTCTGGCGCAGGCTAACCAGGTCCCGCAGAACGTGCTGTCTCTGTTACGTGAATTCTCT

AGATATCCAGCACAGTGGCGGCCGCTCGACACAATATGTATAGGCTACCATGCGAACAAT

TCAACCGACACTGTTGACACAGTACTCGAGAAGAATGTGACAGTGACACACTCTGTTAAC

CTGCTCGAAGACAGCCACAACGGAAAACTATGTAGATTAAAAGGAATAGCCCCACTACAA

TTGGGGAAATGTAACATCGCCGGATGGCTTTTGGGAAACCCAGAATGCGACCCACTGCTT

CCAGTGAGATCATGGTCCTACATTGTAGAAACACCAAACTCTGAGAATGGAATATGTTAT

CCAGGAGATTTCATCGACTATGAGGAGCTGAGGGAGCAATTGAGCTCAGTGTCATCATTC

GAAAGATTCGAAATATTTCCCAAAGAAAGCTCATGGCCCAACCACAACACAAACGGAGTA

ACGGCAGCATGCTCCCATGAGGGGAAAAGCAGTTTTTACAGAAATTTGCTATGGCTGACG

GAGAAGGAGGGCTCATACCCAAAGCTGAAAAATTCTTATGTGAACAAAAAAGGGAAAGAA

GTCCTTGTACTGTGGGGTATTCATCACCCGCCTAACAGTAAGGAACAACAGAATCTCTAT

CAGAATGAAAATGCTTATGTCTCTGTAGTGACTTCAAATTATAACAGGAGATTTACCCCG

GAAATAGCAGAAAGACCCAAAGTAAGAGATCAAGCTGGGAGGATGAACTATTACTGGACC

TTGCTAAAACCCGGAGACACAATAATATTTGAGGCAAATGGAAATCTAATAGCACCAATG

TATGCTTTCGCACTGAGTAGAGGCTTTGGGTCCGGCATCATCACCTCAAACGCATCAATG

CATGAGTGTAACACGAAGTGTCAAACACCCCTGGGAGCTATAAACAGCAGTCTCCCTTAC

CAGAATATACACCCAGTCACAATAGGAGAGTGCCCAAAATACGTCAGGAGTGCCAAATTG

AGGATGGTTACAGGACTAAGGAACATTCCGTCCATTCAATCCAGAGGTCTATTTGGAGCC

ATTGCCGGTTTTATTGAAGGGGGATGGACTGGAATGATAGATGGATGGTATGGTTATCAT

CATCAGAATGAACAGGGATCAGGCTATGCAGCGGATCAAAAAAGCACACAAAATGCCATT

AACGGGATTACAAACAAGGTGAACACTGTTATCGAGAAAATGAACATTCAATTCACAGCT

GTGGGTAAAGAATTCAACAAATTAGAAAAAAGGATGGAAAATTTAAATAAAAAAGTTGAT

GATGGATTTCTGGACATTTGGACATATAATGCAGAATTGTTAGTTCTACTGGAAAATGAA

AGGACTCTGGATTTCCATGACTCAAATGTGAAGAATCTGTATGAGAAAGTAAAAAGCCAA

TTAAAGAATAATGCCAAAGAAATCGGAAATGGATGTTTTGAGTTCTACCACAAGTGTGAC

AATGAATGCATGGAAAGTGTAAGAAATGGGACTTATGATTATCCCAAATATTCAGAAGAG

TCAAAGTTGAACAGGGAAAAGGTAGATGGAGTGAAATTGGAATCAATGGGGATCTATCAG

TAG

SEQ ID NO:165 FOR primer

ATTCTCCGGCGGCCGCTCGACACAATATGTATAGGCTACC

SEQ ID NO:166 REV primer

AGTCTTGCGGCCGCCTATTAATGGTGATGGTGATGATGCTGATAGATCCCCATTGATTCC

SEQ ID NO：167 HA0s.STF2Δ

GACACAATATGTATAGGCTACCATGCGAACAATTCAACCGACACTGTTGACACAGTACTC

GAGAAGAATGTGACAGTGACACACTCTGTTAACCTGCTCGAAGACAGCCACAACGGAAAA

CTATGTAGATTAAAAGGAATAGCCCCACTACAATTGGGGAAATGTAACATCGCCGGATGG

CTTTTGGGAAACCCAGAATGCGACCCACTGCTTCCAGTGAGATCATGGTCCTACATTGTA

GAAACACCAAACTCTGAGAATGGAATATGTTATCCAGGAGATTTCATCGACTATGAGGAG

CTGAGGGAGCAATTGAGCTCAGTGTCATCATTCGAAAGATTCGAAATATTTCCCAAAGAA

AGCTCATGGCCCAACCACAACACAAACGGAGTAACGGCAGCATGCTCCCATGAGGGGAAA

AGCAGTTTTTACAGAAATTTGCTATGGCTGACGGAGAAGGAGGGCTCATACCCAAAGCTG

AAAAATTCTTATGTGAACAAAAAAGGGAAAGAAGTCCTTGTACTGTGGGGTATTCATCAC

CCGCCTAACAGTAAGGAACAACAGAATCTCTATCAGAATGAAAATGCTTATGTCTCTGTA

GTGACTTCAAATTATAACAGGAGATTTACCCCGGAAATAGCAGAAAGACCCAAAGTAAGA

GATCAAGCTGGGAGGATGAACTATTACTGGACCTTGCTAAAACCCGGAGACACAATAATA

TTTGAGGCAAATGGAAATCTAATAGCACCAATGTATGCTTTCGCACTGAGTAGAGGCTTT

GGGTCCGGCATCATCACCTCAAACGCATCAATGCATGAGTGTAACACGAAGTGTCAAACA

CCCCTGGGAGCTATAAACAGCAGTCTCCCTTACCAGAATATACACCCAGTCACAATAGGA

GAGTGCCCAAAATACGTCAGGAGTGCCAAGTTGAGGATGGTTACAGGACTAAGGAACATT

CCGTCCATTCAATCCAGAGGTCTATTTGGAGCCATTGCCGGTTTTATTGAAGGGGGATGG

ACTGGAATGATAGATGGATGGTATGGTTATCATCATCAGAATGAACAGGGATCAGGCTAT

GCAGCGGATCAAAAAAGCACACAAAATGCCATTAACGGGATTACAAACAAGGTGAACACT

GTTATCGAGAAAATGAACATTCAATTCACAGCTGTGGGTAAAGAATTCAACAAATTAGAA

AAAAGGATGGAAAATTTAAATAAAAAAGTTGATGATGGATTTCTGGACATTTGGACATAT

AATGCAGAATTGTTAGTTCTACTGGAAAATGAAAGGACTCTGGATTTCCATGACTCAAAT

GTGAAGAATCTGTATGAGAAAGTAAAAAGCCAATTAAAGAATAATGCCAAAGAAATCGGA

AATGGATGTTTTGAGTTCTACCACAAGTGTGACAATGAATGCATGGAAAGTGTAAGAAAT

GGGACTTATGATTATCCCAAATATTCAGAAGAGTCAAAGTTGAACAGGGAAAAGGTAGAT

GGAGTGAAATTGGAATCAATGGGGATCTATCAGGAATTCTCTAGATATCCAGCACAGTGG

CGGCCGCTCGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTG

AACAAATCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATC

AACAGCGCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATC

AAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACT

GAAGGCGCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAG

TCTGCTAACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAG

CGCCTGAACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTG

GCGCAGGACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATC

GATCTGAAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCATGGAGCG

CCGGTGGATCCTGCTAGCCCATGGACCGAAAACCCGCTGCAGAAAATTGATGCCGCGCTG

GCGCAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTACAAAACCGTTTCAACTCTGCT

ATCACCAACCTGGGCAATACCGTAAACAATCTGTCTGAAGCGCGTAGCCGTATCGAAGAT

TCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCGCAGATTTTGCAGCAGGCCGGT

ACTTCCGTTCTGGCGCAGGCTAACCAGGTCCCGCAGAACGTGCTGTCTCTGTTACGTTAA

TAG

SEQ ID NO:168 FOR primer

AGGCAAGATCTGACACAATATGTATAGGCTACC

SEQ ID NO:169 REV primer

AGTCAGACGCGTCTATTAACGTAACAGAGACAGCAC

SEQ ID NO：170 Ha0s.his

GACACAATATGTATAGGCTACCATGCGAACAATTCAACCGACACTGTTGACACAGTACTC

GAGAAGAATGTGACAGTGACACACTCTGTTAACCTGCTCGAAGACAGCCACAACGGAAAA

CTATGTAGATTAAAAGGAATAGCCCCACTACAATTGGGGAAATGTAACATCGCCGGATGG

CTTTTGGGAAACCCAGAATGCGACCCACTGCTTCCAGTGAGATCATGGTCCTACATTGTA

GAAACACCAAACTCTGAGAATGGAATATGTTATCCAGGAGATTTCATCGACTATGAGGAG

CTGAGGGAGCAATTGAGCTCAGTGTCATCATTCGAAAGATTCGAAATATTTCCCAAAGAA

AGCTCATGGCCCAACCACAACACAAACGGAGTAACGGCAGCATGCTCCCATGAGGGGAAA

AGCAGTTTTTACAGAAATTTGCTATGGCTGACGGAGAAGGAGGGCTCATACCCAAAGCTG

AAAAATTCTTATGTGAACAAAAAAGGGAAAGAAGTCCTTGTACTGTGGGGTATTCATCAC

CCGCCTAACAGTAAGGAACAACAGAATCTCTATCAGAATGAAAATGCTTATGTCTCTGTA

GTGACTTCAAATTATAACAGGAGATTTACCCCGGAAATAGCAGAAAGACCCAAAGTAAGA

GATCAAGCTGGGAGGATGAACTATTACTGGACCTTGCTAAAACCCGGAGACACAATAATA

TTTGAGGCAAATGGAAATCTAATAGCACCAATGTATGCTTTCGCACTGAGTAGAGGCTTT

GGGTCCGGCATCATCACCTCAAACGCATCAATGCATGAGTGTAACACGAAGTGTCAAACA

CCCCTGGGAGCTATAAACAGCAGTCTCCCTTACCAGAATATACACCCAGTCACAATAGGA

GAGTGCCCAAAATACGTCAGGAGTGCCAAATTGAGGATGGTTACAGGACTAAGGAACATT

CCGTCCATTCAATCCAGAGGTCTATTTGGAGCCATTGCCGGTTTTATTGAAGGGGGATGG

ACTGGAATGATAGATGGATGGTATGGTTATCATCATCAGAATGAACAGGGATCAGGCTAT

GCAGCGGATCAAAAAAGCACACAAAATGCCATTAACGGGATTACAAACAAGGTGAACACT

GTTATCGAGAAAATGAACATTCAATTCACAGCTGTGGGTAAAGAATTCAACAAATTAGAA

AAAAGGATGGAAAATTTAAATAAAAAAGTTGATGATGGATTTCTGGACATTTGGACATAT

AATGCAGAATTGTTAGTTCTACTGGAAAATGAAAGGACTCTGGATTTCCATGACTCAAAT

GTGAAGAATCTGTATGAGAAAGTAAAAAGCCAATTAAAGAATAATGCCAAAGAAATCGGA

AATGGATGTTTTGAGTTCTACCACAAGTGTGACAATGAATGCATGGAAAGTGTAAGAAAT

GGGACTTATGATTATCCCAAATATTCAGAAGAGTCAAAGTTGAACAGGGAAAAGGTAGAT

GGAGTGAAATTGGAATCAATGGGGATCTATCAGACGCGTTACCGGTCATCATCACCATCAC

CATTGA

SEQ ID NO:171 FOR primer

ATTCTCCGGCGGCCGCTCGACACAATATGTATAGGCTACC

SEQ ID NO:172 REV primer

AGTCTTGCGGCCGCCTATTAATGGTGATGGTGATGATGCTGATAGATCCCCATTGATTCC

SEQ ID NO：173 HA0s

GACACAATATGTATAGGCTACCATGCGAACAATTCAACCGACACTGTTGACACAGTACTC

GAGAAGAATGTGACAGTGACACACTCTGTTAACCTGCTCGAAGACAGCCACAACGGAAAA

CTATGTAGATTAAAAGGAATAGCCCCACTACAATTGGGGAAATGTAACATCGCCGGATGG

CTTTTGGGAAACCCAGAATGCGACCCACTGCTTCCAGTGAGATCATGGTCCTACATTGTA

GAAACACCAAACTCTGAGAATGGAATATGTTATCCAGGAGATTTCATCGACTATGAGGAG

CTGAGGGAGCAATTGAGCTCAGTGTCATCATTCGAAAGATTCGAAATATTTCCCAAAGAA

AGCTCATGGCCCAACCACAACACAAACGGAGTAACGGCAGCATGCTCCCATGAGGGGAAA

AGCAGTTTTTACAGAAATTTGCTATGGCTGACGGAGAAGGAGGGCTCATACCCAAAGCTG

AAAAATTCTTATGTGAACAAAAAAGGGAAAGAAGTCCTTGTACTGTGGGGTATTCATCAC

CCGCCTAACAGTAAGGAACAACAGAATCTCTATCAGAATGAAAATGCTTATGTCTCTGTA

GTGACTTCAAATTATAACAGGAGATTTACCCCGGAAATAGCAGAAAGACCCAAAGTAAGA

GATCAAGCTGGGAGGATGAACTATTACTGGACCTTGCTAAAACCCGGAGACACAATAATA

TTTGAGGCAAATGGAAATCTAATAGCACCAATGTATGCTTTCGCACTGAGTAGAGGCTTT

GGGTCCGGCATCATCACCTCAAACGCATCAATGCATGAGTGTAACACGAAGTGTCAAACA

CCCCTGGGAGCTATAAACAGCAGTCTCCCTTACCAGAATATACACCCAGTCACAATAGGA

GAGTGCCCAAAATACGTCAGGAGTGCCAAATTGAGGATGGTTACAGGACTAAGGAACATT

CCGTCCATTCAATCCAGAGGTCTATTTGGAGCCATTGCCGGTTTTATTGAAGGGGGATGG

ACTGGAATGATAGATGGATGGTATGGTTATCATCATCAGAATGAACAGGGATCAGGCTAT

GCAGCGGATCAAAAAAGCACACAAAATGCCATTAACGGGATTACAAACAAGGTGAACACT

GTTATCGAGAAAATGAACATTCAATTCACAGCTGTGGGTAAAGAATTCAACAAATTAGAA

AAAAGGATGGAAAATTTAAATAAAAAAGTTGATGATGGATTTCTGGACATTTGGACATAT

AATGCAGAATTGTTAGTTCTACTGGAAAATGAAAGGACTCTGGATTTCCATGACTCAAAT

GTGAAGAATCTGTATGAGAAAGTAAAAAGCCAATTAAAGAATAATGCCAAAGAAATCGGA

AATGGATGTTTTGAGTTCTACCACAAGTGTGACAATGAATGCATGGAAAGTGTAAGAAAT

GGGACTTATGATTATCCCAAATATTCAGAAGAGTCAAAGTTGAACAGGGAAAAGGTAGAT

GGAGTGAAATTGGAATCAATGGGGATCTATCAG

SEQ ID NO:174 FOR primer

AGGCAAGATCTGACACAATATGTATAGGCTACC

SEQ ID NO:175 REV primer

AGTCAGACGCGTCTATTAACGTAACAGAGACAGCAC

SEQ ID NO：176 HA0sHis(PR8)

DTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDSHNGKLCRLKGIAPLQLGKCNIAGWLLGNP

ECDPLLPVRSWSYIVETPNSENGICYPGDFIDYEELREQLSSVSSFERFEIFPKESSWPNHNTNGVTA

ACSHEGKSSFYRNLLWLTEKEGSYPKLKNSYVNKKGKEVLVLWGIHHPPNSKEQQNLYQNENAY

VSVVTSNYNRRFTPEIAERPKVRDQAGRMNYYWTLLKPGDTIIFEANGNLIAPMYAFALSRGFGS

GIITSNASMHECNTKCQTPLGAINSSLPYQNIHPVTIGECPKYVRSAKLRMVTGLRNIPSIQSRGLF

GAIAGFIEGGWTGMIDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNTVIEKMNIQFTAVG

KEFNKLEKRMENLNKKVDDGFLDIWTYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNA

KEIGNGCFEFYHKCDNECMESVRNGTYDYPKYSEESKLNREKVDGVKLESMGIYQHHHHHH

SEQ ID NO：177 STF2Δ.HA0s(PR8)

MAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANRFTANIKGLTQASR

NANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLNEIDRVSGQTQFN

GVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDSLNVHGAPVDPASPWTENPLQKIDAAL

AQVDALRSDLGAVQNRFNSAITNLGNTVNNLSEARSRIEDSDYATEVSNMSRAQILQQAGTSVLA

QANQVPQNVLSLLREFSRYPAQWRPLDTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDSHNG

KLCRLKGIAPLQLGKCNIAGWLLGNPECDPLLPVRSWSYIVETPNSENGICYPGDFIDYEELREQLS

SVSSFERFEIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTEKEGSYPKLKNSYVNKKGKE

VLVLWGIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQAGRMNYYWTLLKP

GDTIIFEANGNLIAPMYAFALSRGFGSGIITSNASMHECNTKCQTPLGAINSSLPYQNIHPVTIGECP

KYVRSAKLRMVTGLRNIPSIQSRGLFGAIAGFIEGGWTGMIDGWYGYHHQNEQGSGYAADQKST

QNAINGITNKVNTVIEKMNIQFTAVGKEFNKLEKRMENLNKKVDDGFLDIWTYNAELLVLLENER

TLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCDNECMESVRNGTYDYPKYSEESKLNR

EKVDGVKLESMGIYQ

SEQ ID NO：178 STF2

ATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAA

TCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGC

GCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGT

CTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGC

GCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCT

AACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTG

AACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAG

GACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTG

AAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCAGAAAGCGTATGAT

GTGAAAGATACAGCAGTAACAACGAAAGCTTATGCCAATAATGGTACTACACTGGATGTA

TCGGGTCTTGATGATGCAGCTATTAAAGCGGCTACGGGTGGTACGAATGGTACGGCTTCT

GTAACCGGTGGTGCGGTTAAATTTGACGCAGATAATAACAAGTACTTTGTTACTATTGGT

GGCTTTACTGGTGCTGATGCCGCCAAAAATGGCGATTATGAAGTTAACGTTGCTACTGAC

GGTACAGTAACCCTTGCGGCTGGCGCAACTAAAACCACAATGCCTGCTGGTGCGACAACT

AAAACAGAAGTACAGGAGTTAAAAGATACACCGGCAGTTGTTTCAGCAGATGCTAAAAAT

GCCTTAATTGCTGGCGGCGTTGACGCTACCGATGCTAATGGCGCTGAGTTGGTCAAAATG

TCTTATACCGATAAAAATGGTAAGACAATTGAAGGCGGTTATGCGCTTAAAGCTGGCGAT

AAGTATTACGCCGCAGATTACGATGAAGCGACAGGAGCAATTAAAGCTAAAACTACAAGT

TATACTGCTGCTGACGGCACTACCAAAACAGCGGCTAACCAACTGGGTGGCGTAGACGGT

AAAACCGAAGTCGTTACTATCGACGGTAAAACCTACAATGCCAGCAAAGCCGCTGGTCAT

GATTTCAAAGCACAACCAGAGCTGGCGGAAGCAGCCGCTAAAACCACCGAAAACCCGCTG

CAGAAAATTGATGCCGCGCTGGCGCAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTA

CAAAACCGTTTCAACTCTGCTATCACCAACCTGGGCAATACCGTAAACAATCTGTCTGAA

GCGCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCG

CAGATTCTGCAGCAGGCCGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTCCCGCAGAAC

GTGCTGTCTCTGTTACGT

SEQ ID No：179 HA1-1his(PR8)

MKFLVNVALVFMVVYISYIYASHNGKLCRLKGIAPLQLGKCNIAGWLLGNPECDPLLPVR

SWSYIVETPNSENGICYPGDFIDYEELREQLSSVSSFERFEIFPKESSWPNHNTNGVTAA

CSHEGKSSFYRNLLWLTEKEGSYPKLKNSYVNKKGKEVLVLWGIHHPPNSKEQQNLYQNE

NAYVSVVTSNYNRRFTPEIAERPKVRDQAGRMNYYWTLLKPGDTIIFEANGNLIAPMYAF

ALSRGFGSGIITSNASMHECNTKCQTPLGAINSSLPYQNIHPVTIGECPKYVRHHHHHH

SEQ ID No：180 HA1-1his(VN)

MKFLVNVALVFMVVYISYIYAEKKHNGKLCDLDGVKPLILRDCSVAGWLLGNPMCDEFIN

VPEWSYIVEKANPVNDLCYPGDFNDYEELKHLLSRINHFEKIQIIPKSSWSSHEASLGVS

SACPYQGKSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTKLYQ

NPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEY

AYKIVKKGDSTIMKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIGECPKYVKHHHHH

H

SEQ ID No：181 HA1-1his(IND)

MKFLVNVALVFMVVYISYIYAEKTHNGKLCDLDGVKPLILRDCSVAGWLLGNPMCDEFIN

VPEWSYIVEKANPTNDLCYPGSFNDYEELKHLLSRINHFEKIQIIPKSSWSDHEASSGVS

SACPYLGSPSFFRNVVWLIKKNSTYPTIKKSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQ

NPTTYISIGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEY

AYKIVKKGDSAIMKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIGECPKYVKHHHHH

H

SEQ ID No：182 HA1-1his(NC)

MKFLVNVALVFMVVYISYIYASHNGKLCLLKGIAPLQLGNCSVAGWILGNPECELLISKE

SWSYIVETPNPENGTCYPGYFADYEELREQLSSVSSFERFEIFPKESSWPNHTVTGVSAS

CSHNGKSSFYRNLLWLTGKNGLYPNLSKSYVNNKEKEVLVLWGVHHPPNIGNQRALYHTE

NAYVSVVSSHYSRRFTPEIAKRPKVRDQEGRINYYWTLLEPGDTIIFEANGNLIAPWYAF

ALSRGFGSGIITSNAPMDECDAKCQTPQGAINSSLPFQNVHPVTIGECPKYVRHHHHHH

SEQ ID No：183 HA1-1(MAL)

ACCACTACCCCAACCAAATCTCACTTCGCAAACCTGAAAGGCACTGAAACCCGTGGCAAG

CTGTGTCCGAAGTGTCTGAACTGCACCGATCTGGACGTCGCACTGGGTCGTCCGAAATGT

ACTGGTAACATTCCGTCCGCGCGTGTCTCCATCCTGCATGAAGTGCGTCCAGTGACCTCC

GGCTGTTTTCCGATTATGCATGATCGTACTAAAATCCGTCAGCTGCCGAACCTGCTGCGT

GGTTACGAACACATTCGTCTGTCCACCCATAACGTTATCAACGCGGAAAACGCGCCGGGC

GGTAGCTATAAAATCGGTACCTCTGGTTCTTGCCCGAACGTGACTAACGGTAACGGCTTC

TTTGCAACCATGGCCTGGGCGGTCCCGAAAAACGACAACAACAAGACCGCGACCAATTCC

CTGACCATCGAAGTCCCGTATATCTGCACCGAAGGTGAAGATCAAATCACGGTTTGGGGC

TTCCACTCCGACAACGAGGCACAAATGGCGAAACTGTACGGTGACAGCAAACCGCAAAAA

TTCACTAGCTCCGCTAACGGTGTTACCACCCACTACGTTTCCCAGATCGGTGGTTTCCCA

AACCAGACCGAAGATGGTGGTCTGCCGCAGTCCGGTCGCATCGTTGTAGATTATATGGTG

CAGAAAAGCGGTAAAACCGGTACCATCACCTACCAGCGTGGCATCCTGCTGCCGCAGAAA

GTTTGGTGCGCTTCCGGTCGTAGCAAAGTAATCAAAGGTTCCCTGCCGCTGATCGGTGAA

GCAGACTGCCTGCACGAGAAATACGGCGGTCTGAACAAAAGCAAGCCGTACTATACCGGC

GAACATGCGAAAGCAATTGGTAACTGTCCAATTTGGGTGAAATAGTAG

SEQ ID No：184 STF2.HA1-1(MAL)

ATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAA

TCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGC

GCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGT

CTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGC

GCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCT

AACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTG

AACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAG

GACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTG

AAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCAGAAAGCGTATGAT

GTGAAAGATACAGCAGTAACAACGAAAGCTTATGCCAATAATGGTACTACACTGGATGTA

TCGGGTCTTGATGATGCAGCTATTAAAGCGGCTACGGGTGGTACGAATGGTACGGCTTCT

GTAACCGGTGGTGCGGTTAAATTTGACGCAGATAATAACAAGTACTTTGTTACTATTGGT

GGCTTTACTGGTGCTGATGCCGCCAAAAATGGCGATTATGAAGTTAACGTTGCTACTGAC

GGTACAGTAACCCTTGCGGCTGGCGCAACTAAAACCACAATGCCTGCTGGTGCGACAACT

AAAACAGAAGTACAGGAGTTAAAAGATACACCGGCAGTTGTTTCAGCAGATGCTAAAAAT

GCCTTAATTGCTGGCGGCGTTGACGCTACCGATGCTAATGGCGCTGAGTTGGTCAAAATG

TCTTATACCGATAAAAATGGTAAGACAATTGAAGGCGGTTATGCGCTTAAAGCTGGCGAT

AAGTATTACGCCGCAGATTACGATGAAGCGACAGGAGCAATTAAAGCTAAAACTACAAGT

TATACTGCTGCTGACGGCACTACCAAAACAGCGGCTAACCAACTGGGTGGCGTAGACGGT

AAAACCGAAGTCGTTACTATCGACGGTAAAACCTACAATGCCAGCAAAGCCGCTGGTCAT

GATTTCAAAGCACAACCAGAGCTGGCGGAAGCAGCCGCTAAAACCACCGAAAACCCGCTG

CAGAAAATTGATGCCGCGCTGGCGCAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTA

CAAAACCGTTTCAACTCTGCTATCACCAACCTGGGCAATACCGTAAACAATCTGTCTGAA

GCGCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCG

CAGATTCTGCAGCAGGCCGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTCCCGCAGAAC

GTGCTGTCTCTGTTAGCGACCACTACCCCAACCAAATCTCACTTCGCAAACCTGAAAGGC

ACTGAAACCCGTGGCAAGCTGTGTCCGAAGTGTCTGAACTGCACCGATCTGGACGTCGCA

CTGGGTCGTCCGAAATGTACTGGTAACATTCCGTCCGCGCGTGTCTCCATCCTGCATGAA

GTGCGTCCAGTGACCTCCGGCTGTTTTCCGATTATGCATGATCGTACTAAAATCCGTCAG

CTGCCGAACCTGCTGCGTGGTTACGAACACATTCGTCTGTCCACCCATAACGTTATCAAC

GCGGAAAACGCGCCGGGCGGTAGCTATAAAATCGGTACCTCTGGTTCTTGCCCGAACGTG

ACTAACGGTAACGGCTTCTTTGCAACCATGGCCTGGGCGGTCCCGAAAAACGACAACAAC

AAGACCGCGACCAATTCCCTGACCATCGAAGTCCCGTATATCTGCACCGAAGGTGAAGAT

CAAATCACGGTTTGGGGCTTCCACTCCGACAACGAGGCACAAATGGCGAAACTGTACGGT

GACAGCAAACCGCAAAAATTCACTAGCTCCGCTAACGGTGTTACCACCCACTACGTTTCC

CAGATCGGTGGTTTCCCAAACCAGACCGAAGATGGTGGTCTGCCGCAGTCCGGTCGCATC

GTTGTAGATTATATGGTGCAGAAAAGCGGTAAAACCGGTACCATCACCTACCAGCGTGGC

ATCCTGCTGCCGCAGAAAGTTTGGTGCGCTTCCGGTCGTAGCAAAGTAATCAAAGGTTCC

CTGCCGCTGATCGGTGAAGCAGACTGCCTGCACGAGAAATACGGCGGTCTGAACAAAAGC

AAGCCGTACTATACCGGCGAACATGCGAAAGCAATTGGTAACTGTCCAATTTGGGTGAAA

TAGTAG

SEQ ID No：185 HA1-2(MAL)

AAAGGCACTGAAACCCGTGGCAAGCTGTGTCCGAAGTGTCTGAACTGCACCGATCTGGAC

GTCGCACTGGGTCGTCCGAAATGTACTGGTAACATTCCGTCCGCGCGTGTCTCCATCCTG

CATGAAGTGCGTCCAGTGACCTCCGGCTGTTTTCCGATTATGCATGATCGTACTAAAATC

CGTCAGCTGCCGAACCTGCTGCGTGGTTACGAACACATTCGTCTGTCCACCCATAACGTT

ATCAACGCGGAAAACGCGCCGGGCGGTAGCTATAAAATCGGTACCTCTGGTTCTTGCCCG

AACGTGACTAACGGTAACGGCTTCTTTGCAACCATGGCCTGGGCGGTCCCGAAAAACGAC

AACAACAAGACCGCGACCAATTCCCTGACCATCGAAGTCCCGTATATCTGCACCGAAGGT

GAAGATCAAATCACGGTTTGGGGCTTCCACTCCGACAACGAGGCACAAATGGCGAAACTG

TACGGTGACAGCAAACCGCAAAAATTCACTAGCTCCGCTAACGGTGTTACCACCCACTAC

GTTTCCCAGATCGGTGGTTTCCCAAACCAGACCGAAGATGGTGGTCTGCCGCAGTCCGGT

CGCATCGTTGTAGATTATATGGTGCAGAAAAGCGGTAAAACCGGTACCATCACCTACCAG

CGTGGCATCCTGCTGCCGCAGAAAGTTTGGTGCGCTTCCGGTCGTAGCAAAGTAATCAAA

GGTTGATAG

SEQ ID No：186 STF2.HA1-2(MAL)

ATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAA

TCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGC

GCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGT

CTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGC

GCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCT

AACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTG

AACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAG

GACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTG

AAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCAGAAAGCGTATGAT

GTGAAAGATACAGCAGTAACAACGAAAGCTTATGCCAATAATGGTACTACACTGGATGTA

TCGGGTCTTGATGATGCAGCTATTAAAGCGGCTACGGGTGGTACGAATGGTACGGCTTCT

GTAACCGGTGGTGCGGTTAAATTTGACGCAGATAATAACAAGTACTTTGTTACTATTGGT

GGCTTTACTGGTGCTGATGCCGCCAAAAATGGCGATTATGAAGTTAACGTTGCTACTGAC

GGTACAGTAACCCTTGCGGCTGGCGCAACTAAAACCACAATGCCTGCTGGTGCGACAACT

AAAACAGAAGTACAGGAGTTAAAAGATACACCGGCAGTTGTTTCAGCAGATGCTAAAAAT

GCCTTAATTGCTGGCGGCGTTGACGCTACCGATGCTAATGGCGCTGAGTTGGTCAAAATG

TCTTATACCGATAAAAATGGTAAGACAATTGAAGGCGGTTATGCGCTTAAAGCTGGCGAT

AAGTATTACGCCGCAGATTACGATGAAGCGACAGGAGCAATTAAAGCTAAAACTACAAGT

TATACTGCTGCTGACGGCACTACCAAAACAGCGGCTAACCAACTGGGTGGCGTAGACGGT

AAAACCGAAGTCGTTACTATCGACGGTAAAACCTACAATGCCAGCAAAGCCGCTGGTCAT

GATTTCAAAGCACAACCAGAGCTGGCGGAAGCAGCCGCTAAAACCACCGAAAACCCGCTG

CAGAAAATTGATGCCGCGCTGGCGCAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTA

CAAAACCGTTTCAACTCTGCTATCACCAACCTGGGCAATACCGTAAACAATCTGTCTGAA

GCGCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCG

CAGATTCTGCAGCAGGCCGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTCCCGCAGAAC

GTGCTGTCTCTGTTAGCGAAAGGCACTGAAACCCGTGGCAAGCTGTGTCCGAAGTGTCTG

AACTGCACCGATCTGGACGTCGCACTGGGTCGTCCGAAATGTACTGGTAACATTCCGTCC

GCGCGTGTCTCCATCCTGCATGAAGTGCGTCCAGTGACCTCCGGCTGTTTTCCGATTATG

CATGATCGTACTAAAATCCGTCAGCTGCCGAACCTGCTGCGTGGTTACGAACACATTCGT

CTGTCCACCCATAACGTTATCAACGCGGAAAACGCGCCGGGCGGTAGCTATAAAATCGGT

ACCTCTGGTTCTTGCCCGAACGTGACTAACGGTAACGGCTTCTTTGCAACCATGGCCTGG

GCGGTCCCGAAAAACGACAACAACAAGACCGCGACCAATTCCCTGACCATCGAAGTCCCG

TATATCTGCACCGAAGGTGAAGATCAAATCACGGTTTGGGGCTTCCACTCCGACAACGAG

GCACAAATGGCGAAACTGTACGGTGACAGCAAACCGCAAAAATTCACTAGCTCCGCTAAC

GGTGTTACCACCCACTACGTTTCCCAGATCGGTGGTTTCCCAAACCAGACCGAAGATGGT

GGTCTGCCGCAGTCCGGTCGCATCGTTGTAGATTATATGGTGCAGAAAAGCGGTAAAACC

GGTACCATCACCTACCAGCGTGGCATCCTGCTGCCGCAGAAAGTTTGGTGCGCTTCCGGT

CGTAGCAAAGTAATCAAAGGTTGATAG

SEQ ID No：187 HA1-2(SH)

AAAGGCACTCGTACCCGCGGTAAGCTGTGCCCGGACTGCCTGAACTGTACCGATCTGGAT

GTTGCACTGGGTCGTCCGATGTGCGTTGGTACCACCCCGTCTGCGAAAGCCAGCATCCTG

CACGAAGTTCGCCCGGTTACTTCCGGTTGTTTCCCGATTATGCATGATCGTACCAAAATT

CGTCAGCTGCCAAACCTGCTGCGTGGCTATGAAAACATTCGTCTGTCCACTCAAAACGTA

ATCGATGCAGAAAAAGCGCTGGGTGGCCCGTATCGTCTGGGTACCAGCGGCTCCTGCCCG

AACGCGACGAGCAAAAGCGGCTTCTTCGCCACCATGGCTTGGGCCGTTCCGAAAGACAAC

AACAAAAACGCTACGAACCCGCTGACCGTCGAAGTCCCGTACATCTGCACCGAAGGCGAA

GATCAGATCACTGTGTGGGGCTTCCACAGCGATGATAAGACCCAGATGAAAAATCTGTAC

GGTGACTCCAACCCGCAGAAATTCACCTCTTCTGCTAACGGTGTAACGACCCACTACGTT

TCTCAGATCGGTGGTTTCCCGGACCAGACGGAAGATGGCGGTCTGCCTCAGTCCGGCCGC

ATCGTAGTTGATTACATGGTCCAGAAACCGGGTAAGACTGGTACCATTGTTTACCAGCGT

GGTGTACTGCTGCCGCAGAAGGTCTGGTGTGCTTCCGGCCGTTCCAAGGTCATTAAGGGC

TGATA

SEQ ID No：188 STF2.HA1-2(SH)

ATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAA

TCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGC

GCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGT

CTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGC

GCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCT

AACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTG

AACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAG

GACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTG

AAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCAGAAAGCGTATGAT

GTGAAAGATACAGCAGTAACAACGAAAGCTTATGCCAATAATGGTACTACACTGGATGTA

TCGGGTCTTGATGATGCAGCTATTAAAGCGGCTACGGGTGGTACGAATGGTACGGCTTCT

GTAACCGGTGGTGCGGTTAAATTTGACGCAGATAATAACAAGTACTTTGTTACTATTGGT

GGCTTTACTGGTGCTGATGCCGCCAAAAATGGCGATTATGAAGTTAACGTTGCTACTGAC

GGTACAGTAACCCTTGCGGCTGGCGCAACTAAAACCACAATGCCTGCTGGTGCGACAACT

AAAACAGAAGTACAGGAGTTAAAAGATACACCGGCAGTTGTTTCAGCAGATGCTAAAAAT

GCCTTAATTGCTGGCGGCGTTGACGCTACCGATGCTAATGGCGCTGAGTTGGTCAAAATG

TCTTATACCGATAAAAATGGTAAGACAATTGAAGGCGGTTATGCGCTTAAAGCTGGCGAT

AAGTATTACGCCGCAGATTACGATGAAGCGACAGGAGCAATTAAAGCTAAAACTACAAGT

TATACTGCTGCTGACGGCACTACCAAAACAGCGGCTAACCAACTGGGTGGCGTAGACGGT

AAAACCGAAGTCGTTACTATCGACGGTAAAACCTACAATGCCAGCAAAGCCGCTGGTCAT

GATTTCAAAGCACAACCAGAGCTGGCGGAAGCAGCCGCTAAAACCACCGAAAACCCGCTG

CAGAAAATTGATGCCGCGCTGGCGCAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTA

CAAAACCGTTTCAACTCTGCTATCACCAACCTGGGCAATACCGTAAACAATCTGTCTGAA

GCGCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCG

CAGATTCTGCAGCAGGCCGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTCCCGCAGAAC

GTGCTGTCTCTGTTAGCGAAAGGCACTCGTACCCGCGGTAAGCTGTGCCCGGACTGCCTG

AACTGTACCGATCTGGATGTTGCACTGGGTCGTCCGATGTGCGTTGGTACCACCCCGTCT

GCGAAAGCCAGCATCCTGCACGAAGTTCGCCCGGTTACTTCCGGTTGTTTCCCGATTATG

CATGATCGTACCAAAATTCGTCAGCTGCCAAACCTGCTGCGTGGCTATGAAAACATTCGT

CTGTCCACTCAAAACGTAATCGATGCAGAAAAAGCGCTGGGTGGCCCGTATCGTCTGGGT

ACCAGCGGCTCCTGCCCGAACGCGACGAGCAAAAGCGGCTTCTTCGCCACCATGGCTTGG

GCCGTTCCGAAAGACAACAACAAAAACGCTACGAACCCGCTGACCGTCGAAGTCCCGTAC

ATCTGCACCGAAGGCGAAGATCAGATCACTGTGTGGGGCTTCCACAGCGATGATAAGACC

CAGATGAAAAATCTGTACGGTGACTCCAACCCGCAGAAATTCACCTCTTCTGCTAACGGT

GTAACGACCCACTACGTTTCTCAGATCGGTGGTTTCCCGGACCAGACGGAAGATGGCGGT

CTGCCTCAGTCCGGCCGCATCGTAGTTGATTACATGGTCCAGAAACCGGGTAAGACTGGT

ACCATTGTTTACCAGCGTGGTGTACTGCTGCCGCAGAAGGTCTGGTGTGCTTCCGGCCGT

TCCAAGGTCATTAAGGGCTGATAG

SEQ ID No:189 HA1-2 (Lee)

AAAGGCACTCAGACCCGTGGCAAGCTGTGTCCGAACTGTTTCAACTGCACCGATCTGGAC

GTTGCACTGGGTCGTCCGAAATGCATGGGTAACATCCCGTCTGCGAAGGTAAGCATCCTG

CACGAAGTTAAACCGGTAACCAGCGGCTGTTTCCCGATCATGCACGACAAAACTAAAATT

CGTCAGCTGCCGAACCTGCTGCGTGGTTATGAGAACATTCGTCTGTCTACCTCTAATGTT

ATCAACGCGGAGACTGCACCAGGTGGCCCATACAAAGTAGGTACCAGCGGTTCCTGTCCG

AACGTTGCGAATCGTAACGGCTTCTTCAACACTATGGCGTGGGTTATCCCGAAAGATAAC

AATAAAACTGCAATTAACCCGGTAACTGTAGAAGTTCCGTACATCTGCTCCGAAGGCGAG

GACCAGATTACGGTATGGGGCTTTCACAGCGACGATAAAACCCAGATGGAGCGTCTGTAC

GGTGACTCTAACCCGCAGAAATTCACCTCCTCCGCGAACGGCGTTACCACCCACTATGTT

TCTCAGATCGGCGGTTTCCCGAATCAGACCGAAGACGAAGGCCTGAAGCAGTCCGGCCGT

ATTGTTGTAGACTACATGGTTCAGAAGCCGGGCAAAACTGGTACCATTGTATACCAGCGC

GGCATCCTGCTGCCGCAGAAAGTTTGGTGCGCTTCCGGTCGTAGCAAAGTAATCAAAGGT

TGATAG

SEQ ID No:190 STF2.HA1-2 (Lee)

ATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAA

TCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGC

GCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGT

CTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGC

GCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCT

AACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTG

AACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAG

GACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTG

AAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCAGAAAGCGTATGAT

GTGAAAGATACAGCAGTAACAACGAAAGCTTATGCCAATAATGGTACTACACTGGATGTA

TCGGGTCTTGATGATGCAGCTATTAAAGCGGCTACGGGTGGTACGAATGGTACGGCTTCT

GTAACCGGTGGTGCGGTTAAATTTGACGCAGATAATAACAAGTACTTTGTTACTATTGGT

GGCTTTACTGGTGCTGATGCCGCCAAAAATGGCGATTATGAAGTTAACGTTGCTACTGAC

GGTACAGTAACCCTTGCGGCTGGCGCAACTAAAACCACAATGCCTGCTGGTGCGACAACT

AAAACAGAAGTACAGGAGTTAAAAGATACACCGGCAGTTGTTTCAGCAGATGCTAAAAAT

GCCTTAATTGCTGGCGGCGTTGACGCTACCGATGCTAATGGCGCTGAGTTGGTCAAAATG

TCTTATACCGATAAAAATGGTAAGACAATTGAAGGCGGTTATGCGCTTAAAGCTGGCGAT

AAGTATTACGCCGCAGATTACGATGAAGCGACAGGAGCAATTAAAGCTAAAACTACAAGT

TATACTGCTGCTGACGGCACTACCAAAACAGCGGCTAACCAACTGGGTGGCGTAGACGGT

AAAACCGAAGTCGTTACTATCGACGGTAAAACCTACAATGCCAGCAAAGCCGCTGGTCAT

GATTTCAAAGCACAACCAGAGCTGGCGGAAGCAGCCGCTAAAACCACCGAAAACCCGCTG

CAGAAAATTGATGCCGCGCTGGCGCAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTA

CAAAACCGTTTCAACTCTGCTATCACCAACCTGGGCAATACCGTAAACAATCTGTCTGAA

GCGCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCG

CAGATTCTGCAGCAGGCCGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTCCCGCAGAAC

GTGCTGTCTCTGTTAGCGAAAGGCACTCAGACCCGTGGCAAGCTGTGTCCGAACTGTTTC

AACTGCACCGATCTGGACGTTGCACTGGGTCGTCCGAAATGCATGGGTAACATCCCGTCT

GCGAAGGTAAGCATCCTGCACGAAGTTAAACCGGTAACCAGCGGCTGTTTCCCGATCATG

CACGACAAAACTAAAATTCGTCAGCTGCCGAACCTGCTGCGTGGTTATGAGAACATTCGT

CTGTCTACCTCTAATGTTATCAACGCGGAGACTGCACCAGGTGGCCCATACAAAGTAGGT

ACCAGCGGTTCCTGTCCGAACGTTGCGAATCGTAACGGCTTCTTCAACACTATGGCGTGG

GTTATCCCGAAAGATAACAATAAAACTGCAATTAACCCGGTAACTGTAGAAGTTCCGTAC

ATCTGCTCCGAAGGCGAGGACCAGATTACGGTATGGGGCTTTCACAGCGACGATAAAACC

CAGATGGAGCGTCTGTACGGTGACTCTAACCCGCAGAAATTCACCTCCTCCGCGAACGGC

GTTACCACCCACTATGTTTCTCAGATCGGCGGTTTCCCGAATCAGACCGAAGACGAAGGC

CTGAAGCAGTCCGGCCGTATTGTTGTAGACTACATGGTTCAGAAGCCGGGCAAAACTGGT

ACCATTGTATACCAGCGCGGCATCCTGCTGCCGCAGAAAGTTTGGTGCGCTTCCGGTCGT

AGCAAAGTAATCAAAGGTTGATAG

SEQ ID No:191 HA1-2 (Ohio)

AAAGGCACTAAAACCCGTGGCAAGCTGTGTCCGAAGTGTCTGAACTGCACCGATCTGGAC

GTCGCACTGGGTCGTCCGAAATGTACTGGTAACATTCCGTCCGCGGAAGTCTCCATCCTG

CATGAAGTGCGTCCAGTGACCTCCGGCTGTTTTCCGATTATGCATGATCGTACTAAAATC

CGTCAGCTGCCGAACCTGCTGCGTGGTTACGAACACATTCGTCTGTCCACCCATAACGTT

ATCAACGCGGAAAAGGCGCCGGGCGGTCCCTATAAAATCGGTACCTCTGGTTCTTGCCCG

AACGTGACTAACGGTAACGGCTTCTTTGCAACCATGGCCTGGGCGGTCCCGAAAAACGAC

AACAACAAGACCGCGACCAATTCCCTGACCATCGAAGTCCCGTATATCTGCACCGAAGGT

GAAGATCAAATCACGATTTGGGGCTTCCACTCCGACAGCGAGACACAAATGGCGAAACTG

TACGGTGACAGCAAACCGCAAAAATTCACTAGCTCCGCTAACGGTGTTACCACCCACTAC

GTTTCCCAGATCGGTGGTTTCCCAAACCAGACCGAAGATGGTGGTCTGCCGCAGTCCGGT

CGCATCGTTGTAGATTATATGGTGCAGAAAAGCGGTAAAACCGGTACCATCACCTACCAG

CGTGGCATCCTGCTGCCGCAGAAAGTTTGGTGCGCTTCCGGTCGTAGCAAAGTAATCAAA

GGTTGATAG

SEQ ID No:192 STF2.HA1-2 (Ohio)

ATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAA

TCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGC

GCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGT

CTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGC

GCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCT

AACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTG

AACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAG

GACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTG

AAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCAGAAAGCGTATGAT

GTGAAAGATACAGCAGTAACAACGAAAGCTTATGCCAATAATGGTACTACACTGGATGTA

TCGGGTCTTGATGATGCAGCTATTAAAGCGGCTACGGGTGGTACGAATGGTACGGCTTCT

GTAACCGGTGGTGCGGTTAAATTTGACGCAGATAATAACAAGTACTTTGTTACTATTGGT

GGCTTTACTGGTGCTGATGCCGCCAAAAATGGCGATTATGAAGTTAACGTTGCTACTGAC

GGTACAGTAACCCTTGCGGCTGGCGCAACTAAAACCACAATGCCTGCTGGTGCGACAACT

AAAACAGAAGTACAGGAGTTAAAAGATACACCGGCAGTTGTTTCAGCAGATGCTAAAAAT

GCCTTAATTGCTGGCGGCGTTGACGCTACCGATGCTAATGGCGCTGAGTTGGTCAAAATG

TCTTATACCGATAAAAATGGTAAGACAATTGAAGGCGGTTATGCGCTTAAAGCTGGCGAT

AAGTATTACGCCGCAGATTACGATGAAGCGACAGGAGCAATTAAAGCTAAAACTACAAGT

TATACTGCTGCTGACGGCACTACCAAAACAGCGGCTAACCAACTGGGTGGCGTAGACGGT

AAAACCGAAGTCGTTACTATCGACGGTAAAACCTACAATGCCAGCAAAGCCGCTGGTCAT

GATTTCAAAGCACAACCAGAGCTGGCGGAAGCAGCCGCTAAAACCACCGAAAACCCGCTG

CAGAAAATTGATGCCGCGCTGGCGCAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTA

CAAAACCGTTTCAACTCTGCTATCACCAACCTGGGCAATACCGTAAACAATCTGTCTGAA

GCGCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCG

CAGATTCTGCAGCAGGCCGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTCCCGCAGAAC

GTGCTGTCTCTGTTAGCGAAAGGCACTAAAACCCGTGGCAAGCTGTGTCCGAAGTGTCTG

AACTGCACCGATCTGGACGTCGCACTGGGTCGTCCGAAATGTACTGGTAACATTCCGTCC

GCGGAAGTCTCCATCCTGCATGAAGTGCGTCCAGTGACCTCCGGCTGTTTTCCGATTATG

CATGATCGTACTAAAATCCGTCAGCTGCCGAACCTGCTGCGTGGTTACGAACACATTCGT

CTGTCCACCCATAACGTTATCAACGCGGAAAAGGCGCCGGGCGGTCCCTATAAAATCGGT

ACCTCTGGTTCTTGCCCGAACGTGACTAACGGTAACGGCTTCTTTGCAACCATGGCCTGG

GCGGTCCCGAAAAACGACAACAACAAGACCGCGACCAATTCCCTGACCATCGAAGTCCCG

TATATCTGCACCGAAGGTGAAGATCAAATCACGATTTGGGGCTTCCACTCCGACAGCGAG

ACACAAATGGCGAAACTGTACGGTGACAGCAAACCGCAAAAATTCACTAGCTCCGCTAAC

GGTGTTACCACCCACTACGTTTCCCAGATCGGTGGTTTCCCAAACCAGACCGAAGATGGT

GGTCTGCCGCAGTCCGGTCGCATCGTTGTAGATTATATGGTGCAGAAAAGCGGTAAAACC

GGTACCATCACCTACCAGCGTGGCATCCTGCTGCCGCAGAAAGTTTGGTGCGCTTCCGGT

CGTAGCAAAGTAATCAAAGGTTGATAG

SEQ ID No:193 FOR primer

CGGATAACAATTCCCCTCTAG

SEQ ID No:194 REV primer

CGAAGTGAGATTTGGTTGGAGTAGTGGTCGCTAACAGAGACAGCACGTTC

SEQ ID No:195 FOR primer

CCCGCAGAACGTGCTGTCTCTGTTAGCGACCACTACCCCAACCAAATCTC

SEQ ID No:196 REV primer

CTATTTCACCCAAATTGGAC

SEQ ID No:197 REV primer

CACAGCTTGCCACGGGTTTCAGTGCCTTTCGCTAACAGAGACAGCACGTTC

SEQ ID No:198 FOR primer

CCCGCAGAACGTGCTGTCTCTGTTAGCGAAAGGCACTGAAACCCGTGGC

SEQ ID No:199 REV primer

GACTAGACGCTCAGCTATCAACCTTTGATTACTTTGCTACGACC

SEQ ID No:200 REV primer

CACAGCTTACCGCGGGTACGAGTGCCTTTCGCTAACAGAGACAGCACGTTC

SEQ ID No:201 FOR primer

CGGGATCCAAAGGCACTCGTACCCGCGGTAAG

SEQ ID No:202 REV primer

GACTAGACGCTCAGCTATCAGCCCTTAATGACCTTGGAACGGCC

SEQ ID No:203 REV primer

GCATAGTTTTCCTCTGGTCTGTGTTCCTTTCGCTAACAGAGACAGCACGTTC

SEQ ID No:204 FOR primer

AAAGGAACACAGACCAGAGG

SEQ ID No:205 REV primer

CTACTACCCTTTTATTACCTTGCTCC

SEQ ID No:206 REV primer

CACAGCTTGCCACGGGTTTTAGTGCCTTTCGCTAACAGAGACAGCACGTTC

SEQ ID No:207 FOR primer

AAAGGCACTAAAACCCGTGGC

SEQ ID No:208 REV primer

GACTAGACGCTCAGCTATCAACCTTTGATTACTTTGCTACGACC

SEQ ID NO：209 STF2.HA1-1(MAL)

MAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANRFTANIKG

LTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRL

NEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDSLNVQKAYD

VKDTAVTTKAYANNGTTLDVSGLDDAAIKAATGGTNGTASVTGGAVKFDADNNKYFVTIG

GFTGADAAKNGDYEVNVATDGTVTLAAGATKTTMPAGATTKTEVQELKDTPAVVSADAKN

ALIAGGVDATDANGAELVKMSYTDKNGKTIEGGYALKAGDKYYAADYDEATGAIKAKTTS

YTAADGTTKTAANQLGGVDGKTEVVTIDGKTYNASKAAGHDFKAQPELAEAAAKTTENPL

QKIDAALAQVDALRSDLGAVQNRFNSAITNLGNTVNNLSEARSRIEDSDYATEVSNMSRA

QILQQAGTSVLAQANQVPQNVLSLLATTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVA

LGRPKCTGNIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVIN

AENAPGGSYKIGTSGSCPNVTNGNGFFATMAWAVPKNDNNKTATNSLTIEVPYICTEGED

QITVWGFHSDNEAQMAKLYGDSKPQKFTSSANGVTTHYVSQIGGFPNQTEDGGLPQSGRI

VVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKS

KPYYTGEHAKAIGNCPIWVK

SEQ ID NO：210 STF2.HA1-2(MAL)

MAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANRFTANIKG

LTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRL

NEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDSLNVQKAYD

VKDTAVTTKAYANNGTTLDVSGLDDAAIKAATGGTNGTASVTGGAVKFDADNNKYFVTIG

GFTGADAAKNGDYEVNVATDGTVTLAAGATKTTMPAGATTKTEVQELKDTPAVVSADAKN

ALIAGGVDATDANGAELVKMSYTDKNGKTIEGGYALKAGDKYYAADYDEATGAIKAKTTS

YTAADGTTKTAANQLGGVDGKTEVVTIDGKTYNASKAAGHDFKAQPELAEAAAKTTENPL

QKIDAALAQVDALRSDLGAVQNRFNSAITNLGNTVNNLSEARSRIEDSDYATEVSNMSRA

QILQQAGTSVLAQANQVPQNVLSLLAKGTETRGKLCPKCLNCTDLDVALGRPKCTGNIPS

ARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGSYKIG

TSGSCPNVTNGNGFFATMAWAVPKNDNNKTATNSLTIEVPYICTEGEDQITVWGFHSDNE

AQMAKLYGDSKPQKFTSSANGVTTHYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKT

GTITYQRGILLPQKVWCASGRSKVIKG

SEQ ID NO：211 STF2.HA1-2(SH)

MAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANRFTANIKG

LTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRL

NEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDSLNVQKAYD

VKDTAVTTKAYANNGTTLDVSGLDDAAIKAATGGTNGTASVTGGAVKFDADNNKYFVTIG

GFTGADAAKNGDYEVNVATDGTVTLAAGATKTTMPAGATTKTEVQELKDTPAVVSADAKN

ALIAGGVDATDANGAELVKMSYTDKNGKTIEGGYALKAGDKYYAADYDEATGAIKAKTTS

YTAADGTTKTAANQLGGVDGKTEVVTIDGKTYNASKAAGHDFKAQPELAEAAAKTTENPL

QKIDAALAQVDALRSDLGAVQNRFNSAITNLGNTVNNLSEARSRIEDSDYATEVSNMSRA

QILQQAGTSVLAQANQVPQNVLSLLAKGTRTRGKLCPDCLNCTDLDVALGRPMCVGTTPS

AKASILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYENIRLSTQNVIDAEKALGGPYRLG

TSGSCPNATSKSGFFATMAWAVPKDNNKNATNPLTVEVPYICTEGEDQITVWGFHSDDKT

QMKNLYGDSNPQKFTSSANGVTTHYVSQIGGFPDQTEDGGLPQSGRIVVDYMVQKPGKTG

TIVYQRGVLLPQKVWCASGRSKVIKG

SEQ ID NO：212 STF2

ATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAA

TCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGC

GCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGT

CTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGC

GCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCT

AACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTG

AACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAG

GACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTG

AAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCAGAAAGCGTATGAT

GTGAAAGATACAGCAGTAACAACGAAAGCTTATGCCAATAATGGTACTACACTGGATGTA

TCGGGTCTTGATGATGCAGCTATTAAAGCGGCTACGGGTGGTACGAATGGTACGGCTTCT

GTAACCGGTGGTGCGGTTAAATTTGACGCAGATAATAACAAGTACTTTGTTACTATTGGT

GGCTTTACTGGTGCTGATGCCGCCAAAAATGGCGATTATGAAGTTAACGTTGCTACTGAC

GGTACAGTAACCCTTGCGGCTGGCGCAACTAAAACCACAATGCCTGCTGGTGCGACAACT

AAAACAGAAGTACAGGAGTTAAAAGATACACCGGCAGTTGTTTCAGCAGATGCTAAAAAT

GCCTTAATTGCTGGCGGCGTTGACGCTACCGATGCTAATGGCGCTGAGTTGGTCAAAATG

TCTTATACCGATAAAAATGGTAAGACAATTGAAGGCGGTTATGCGCTTAAAGCTGGCGAT

AAGTATTACGCCGCAGATTACGATGAAGCGACAGGAGCAATTAAAGCTAAAACTACAAGT

TATACTGCTGCTGACGGCACTACCAAAACAGCGGCTAACCAACTGGGTGGCGTAGACGGT

AAAACCGAAGTCGTTACTATCGACGGTAAAACCTACAATGCCAGCAAAGCCGCTGGTCAT

GATTTCAAAGCACAACCAGAGCTGGCGGAAGCAGCCGCTAAAACCACCGAAAACCCGCTG

CAGAAAATTGATGCCGCGCTGGCGCAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTA

CAAAACCGTTTCAACTCTGCTATCACCAACCTGGGCAATACCGTAAACAATCTGTCTGAA

GCGCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCG

CAGATTCTGCAGCAGGCCGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTCCCGCAGAAC

GTGCTGTCTCTGTTACGT

SEQ ID NO:213 B/ chevron/16/18HA

MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLK

GTKTRGKLCPNCLNCTDLDVALGRPMCMGTIPSAKASILHEVRPVTSGCFPIMHDRTKIR

QLPNLLRGYENIRLSTHNVINAERAPGGPYRLGTSGSCPNVTSRNGFFATMAWAVPRDNK

TATNPLTVEVPYICTKGEDQITVWGFHSDDKTQMKNLYGDSNPQKFTSSANGVTTHYVSQ

IGDFPNQTEDGGLPQSGRIVVDYMVQKPGKTGTIVYQRGVLLPQKVWCASGRSKVIKGSL

PLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKER

GFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEV

KNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELAVLLSNEGIINSEDEHLLALER

KLKKMLGPSAVDIGNGCFETKHKCNQTCLDRIAAGTFNAGEFSLPTFDSLNITAASLNDD

GLDNHTILLYYSTAASSLAVTLMIAIFIVYMVSRDNVSCSICL

SEQ ID NO:787 B/ Victoria/2/87HA

MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLK

GTKTRGKLCPKCLNCTDLDVALGRPKCTGTIPSAKASILHEVKPVTSGCFPIMHDRTKIR

QLPNLLRGYEHIRLSTHNVINAETAPGGPYKVGTSGSCPNVTNGNGFFATMAWAVPKNDN

NKTATNPLTVEVPYICTEGEDQITVWGFHSDNEAQMVKLYGDSKPQKFTSSANGVTTHYV

SQIGGFPNQAEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKG

SLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLK

EKGFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSEL

EVKNLQRLSGAMDELHNKILELDEKVDDLRADTISSQIELAVLLSNEGIINSEDEHLLAL

ERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFNAGEFSLPTFDSLNITAASLN

DDGLDNHTILLYYSTAASSLAVTLMIAIFIVYMVSRDNVSCSICL

Embodiment 13: have the flagellin for the artificial transformation cysteine residues of chemical conjugation

The crystal structure of bacterial flagellin salmonella typhimurium flagellin the 2nd type (STF2, SEQ ID NO:312) is by report (people such as Mi Cang, nature 424,643-650 (2003)).Reported complete atomic model, and carried out the activatory detailed structure-functional study (Smith, people such as K.D., natural immunity 4:1247-1253 (2003)) of TLR5 based on this structure by the bacterial flagellum filamentous of the freezing microscopy of electronics.Clock sample receptor 5 (TLR5) identification is positioned on the flagellin, for the intrinsic site (natural immunity 4 (12): 1247-1253) that precursor forms and the antibacterial motion is required.Mutation analysis proves, the TLR5 activity of flagellin is present in two zones in N-and the terminal functional domain of C-, is respectively to contain 39 and 31 amino acid whose elongations people such as () Smiths.These zones, zone for emphasizing with Lycoperdon polymorphum Vitt among Figure 18.These regional alanine-scannings cause change, identify the activatory sudden change of many TLR5 of attenuating, but do not have a single sudden change elimination activity fully, hint the pliability or the redundancy that in the TLR5 binding site, have to a certain degree.This facilitates the possible notion that develops, or not so antibacterial may develop easily and detected by TLR5 to escape.Lacking the hypermutation hinge region and keep inherent N-and the terminal functional domain of C-, produce a kind of active protein of complete TLR5 (STF2 Δ) of still possessing, prove the terminal functional domain (jointly) of N-and C-, is necessary and sufficient for the TLR5 activation.

Lysine residue is for being used for various chemical constitution chemical conjugations extremely, such as the substrate that makes things convenient for of protein carriers such as flagellin.In 30 lysine residues of STF2 (SEQ ID NO:312), have only one to be positioned at the TLR5 activation site of defining through experiment, and this special lysine is in all bacterial flagellins and extrinsic (Smith, people such as K.D., natural immunity 4:1247-1253 (2003)).In all the other 29 lysines, 24 hypervariable regions that are arranged in flagellin are arranged, therefore remaining 5 lysine residues in STF2 Δ (SEQ ID NO:313).Structure among Figure 19 and 20 shows, most of lysines spatially are away from TLR5 mobilizing function territory, therefore as if lysine can be at random and peptide or carbohydrate antigen conjugation, and reasonably may not can disturb the proteic TLR5 activity of conjugation STF2 Δ (SEQ ID NO:313) that become.As if five lysine positions quite near TLR5 activation site; If when the conjugation of antigen and lysine can influence the TLR5 activity, these residues will at first be considered and suddenly change.

Can conjugated additionally site (for example cysteine or extra lysine) will be supplied, by this type of residue being inserted in hypervariable region or the afterbody, and transform to total length (STF2 (SEQ ID NO:312)) or STF2 Δ (SEQ ID NO:313) with artificial apart from long-range intrinsic N-of TLR5 binding site and the terminal functional domain of C-.Also exist and be used for conjugation to other amino acid whose chemical method.These comprise carboxyamino acid (glutamic acid, aspartic acid) and carboxyl terminal aminoacid, arginine, histidine, tryptophan, tyrosine and serine.Arbitrary these strategies can be used for the antigenic structure conjugation to flagellin, and do not disturb TLR5 activation site.

Some type antigen comprises polysaccharide, can not carry out reorganization fusion dna technology, also can not bestow synthetic chemistry of peptides, and therefore can't be with gene or the synthetic method binding part to clock sample receptor (TLR).In addition, the gene of peptide class and TLR part or synthesis type merge, and may suppress the suitably folding of TLR part.Can utilize the chemical conjugation of antigen and TLR part (through folding and purification).The chemical conjugation of peptide class also allows to make antigen to be positioned over the special site of TLR part, therefore limits the interference to the receptors bind of TLR part, or makes the exposure maximization to antigen receptor.The conjugation of many peptide class antigens and TLR part, also can by increase with antigen represent to immune configuration heterogeneity, exempt from different originality and maximize and make.For being provided for the site that this type of antigen and flagellin carry out chemical conjugation, satisfying cysteine residues is transformed in the coding STF2 Δ (flagellin that lacked of hypermutation hinge region wherein; SEQ ID NO:313 and SEQ ID NO:314) gene in different loci.

Materials and methods

Polymerase chain reaction (PCR): (Ka Sibeide (Carlsbad) is to be used for the pcr amplification that motion that all explanations of using following manufacturers are as the criterion is carried out CA) to the high correctness test kit of PCR SuperMix for catalog number 12532-016, Yin Weituo King Company.

1. following composition is added in each reaction tube according to random order:

A.45 μ l

The high correctness of PCR SuperMix (PCR reaction buffer)

B. primer solution (each primer of 10pMol final concentration)

C. template DNA solution (10ng plasmid DNA)

2. reaction volume is adjusted to 50 μ l with water

3. test tube is added a cover and is loaded in the thermo cycler

4. test tube is cultivated in 94 ℃ times 30 to 120 seconds, so that complete degeneration of template and activating enzymes

5. carry out following pcr amplification and reach 25-35 circulation:

A. under 94 ℃, carry out degeneration 15-30 second

B. under 50-55 ℃, bind 15-30 second

C. under 68-72 ℃, prolong the PCR product size of 1 minute every kb

D. if need, be cooled to 4 ℃ and keep up to next processing procedure required

The structure of STF2 Δ gene (SEQ ID NO:314): the total length flagellin fljb of salmonella typhimurium (STF2, SEQ ID NO:312) is coded by 1.5kb gene (SEQ ID NO:315).For producing STF2 Δ (SEQ ID NO:314), then will be corresponding to sequence (amino acid/11 70 to 415 of the SEQ ID NO:312) disappearance of hypermutation hinge region, and displacement is necessary to help through design for the TLR5 citation, the NH2 of STF2 (SEQ ID NO:315) and the effect (Smith of COOH end sequence, K.D. wait the people, 2004) the flexibly connecting of weak point, (GAPVDPASPW (SEQ ID NO:336)).Clock sample receptor 5 (TLR5) identification is positioned on the flagellin, for the intrinsic site (natural immunity 4 (12): 1247-1253) that precursor forms and the antibacterial motion is required.

For producing STF2 Δ plasmid, use two step PCR (Figure 21) then.In first reaction, be with the coding STF2.OVA (fusion of total length STF2 and total length ovalbumin, SEQ ID NO:316) plasmid, be mixed in the PCR reaction buffer with primer STF28BGF-1 (SEQ ID NO:317) and STF28MCR-1 (SEQ ID NO:318), and mixture is increased in aforesaid PCR reaction.In parallel reaction, be that STF2.OVA template plasmid and primer STF28.MCF-2 (SEQ ID NO:319) and STF28ECR-2 (SEQ ID NO:320) are mixed in the PCR reaction buffer, and mixture is increased in aforesaid PCR reaction.

Described pcr amplification reaction produces respectively～500bp and 270bp fragment.These PCR products and primer STF28BGF-1 (SEQ ID NO:317) and STF28ECR-2 (SEQ ID NO:320) are mixed in the PCR reaction buffer, and mixture is increased in aforesaid PCR reaction.With from then on the reaction increase DNA product (770bp), decomposed 2 hours down in 37 ℃ with BglII and EcoRI restriction endonuclease, and be engaged in advance through BglII and EcoRI digestion and, pMTBiP/V5-His B (Yin Weituo King Company with calf intestinal alkaline phosphatase (CIP) processing, Ka Sibeide, CA) in.Get equal portions joint mixture and be used for transformed into escherichia coli TOP10 cell.The construct that is become is used to produce cysteine modified STF2 Δ construct.

Structure is used to express the STF2 Δ gene of single cysteine residues through transformation: for cysteine residues being directed in the STF2 polypeptide allocation really, use then the pMT/STF2 Δ as with primer as described below to carrying out the template among the PCR.

For making up STF2 Δ .3 ' Cys (SEQ ID NO:325) plasmid, satisfy pMT/STF2 Δ plasmid and 3 ' forward 1 (SEQ ID NO:321) and 3 ' reverse 1 (SEQ ID NO:322) primer are mixed in the PCR reaction buffer, and mixture is increased in aforesaid PCR reacts.

For making up 5 ' Cys.STF2 Δ (SEQ ID NO:326) plasmid, satisfy pMT/STF2 Δ plasmid and 5 ' forward 2 (SEQ ID NO:323) and 5 ' reverse 2 (SEQ ID NO:324) primer are mixed in the PCR reaction buffer, and mixture is increased in aforesaid PCR reacts.

For STF2 Δ .3 ' Cys (SEQ ID NO:325) and 5 ' Cys.STF2 Δ (SEQ ID NO:326) construct, be that the PCR product that will be produced decomposes with Nde1 and Blp1 restriction endonuclease, and by agargel electrophoresis art purification.Purified fragment is bonded to via compatible termination, and (the Nova gold, horse ground is gloomy, WI) plasmid DNA through the pET24a of Nde1 and decomposition of Blp1 restriction endonuclease and CIP processing in advance.The construct that is become is called after pET/STF2 Δ .3 ' Cys and pET/5 ' Cys.STF2 Δ respectively.

For making up the STF2 Δ. hinge Cys (SEQ ID NO:331) plasmid, satisfy utilization and be similar to the described two step PCR of Figure 18.By pMT/STF2 Δ and paired hinge forward 1 (SEQ ID NO:327) and hinge reverse 3 (SEQ ID NO:328) primer are mixed in the PCR reaction buffer, and mixture is increased in aforesaid PCR reaction, and produce a fragment.Another fragment is by pMT/STF2 Δ and paired hinge forward 2 (SEQ ID NO:329) and hinge reverse 4 (SEQ ID NO:330) primer are mixed in the PCR reaction buffer, and mixture increased in aforesaid PCR reaction and produces.To derive from the equal portions combination of each these PCR reaction, and be mixed in the PCR reaction buffer, and mixture is increased in aforesaid PCR reaction with hinge forward 1 (SEQ ID NO:327) and hinge reverse 4 (SEQ ID NO:330) primer.Final PCR product via agargel electrophoresis art purification, and is decomposed with Nde1 and Blp1 restriction endonuclease, and purified fragment is engaged in the foregoing pET24a carrier.The construct that is become is a called after pET/STF2 Δ. hinge Cys.

Protein expression: with plasmid pET/STF2 Δ .3 ' Cys, pET/5 ' Cys.STF2 Δ and pET/STF2 Δ. hinge Cys such as following being converted in competent escherichia coli BLR (DE3) cell.BLR (DE3) cell and the 1 μ l plasmid DNA (1 μ g/ μ l) of 20 μ l equal portions are added to, in the 1.5-ml of pre-cooling on ice bullet lid polypropylene test tube, and with mixture cultivation 30 minutes.Test tube is not added certain 30 seconds of agitating heating in 42 ℃ of water-baths, placed then on ice again 2 minutes in addition.The room temperature SOC of 250 μ l is added to the cell of each pipe through heat shock.

Cell (in 250rpm vibration down) under 37 ℃ was recovered 60 minutes, coat afterwards on the selection culture medium that contains kanamycin.Be coated with the transformation mixture of various different equal portions, and plate was cultivated 18 hours down in 37 ℃.Picking colony also is seeded in Luria-Bertani (LB) fluid medium that replenishes with 25 μ g/ml kanamycin and 12.5 μ g/ml tetracyclines, and overnight incubation.By fresh LB culture is inoculated in the overnight culture 1:100 of equal portions dilution, and under 37 ℃, follow vibration to cultivate.OD when culture ₆₀₀Arrive at 0.6 to 1.0 o'clock, express for the 1mM induced protein by adding isopropylthio-(IPTG) to final concentration.After inducing a few hours, collect cell for expressing by the SDS-PAGE analysing protein.Replenish with the culture the LB of 0.5% glucose, 25 μ g/ml kanamycin and 12.5 μ g/ml tetracyclines from growing in, after adding glycerol (7% final concentration), be frozen in-80 ℃, and be prepared into the glycerol reserve.

Protein purification: protein S TF2 Δ .3 ' Cys (SEQ ID NO:332) and STF2 Δ. expression and the purification of hinge Cys (SEQ ID NO:333) are as follows.The glycerol reserve that will have escherichia coli BLR (DE3) cell of desired plasmid, inoculation are gone into 10L and are contained in vibration-flask of LB, and follow constant vibration to cultivate under 37 ℃.When culture arrives A ₆₀₀During=0.8 optical density (OD), express by adding 1mM IPTG induced protein, and under 37 ℃, follow constant vibration to cultivate 4 hours culture, gather in the crops afterwards.By in 5,000rpm (SLA3000 turns) is low-speed centrifugal 10 minutes down, and from 10L culture collection cell, and being suspended in 50mM Tris-HCl pH 8.0,1mM EDTA is among the 1mM DTT (100ml/10L).

By with cell suspending liquid in 18, under the 000psi, destroy cell by little liquefier twice.By in 10,000rpm (SS34 turns) centrifugal 15 minutes down, and insolubility material and soluble protein are separated.Under described condition of culture, at the same level part of all STF2 Δ protein and insolubility material, and be formed at and be the stable inclusion body that solid sediment is collected after centrifugal.With 50mM Tris-HCl, pH 8.0 with inclusion body, and 0.1M NaCl and 0.5% Triton X-100 clean twice, wash twice with the same buffer that does not contain cleaning agent subsequently.Each cleaning all uses the dounce homogenizer to carry out, and passes through in 10, and 000rpm (SS34 turns) centrifugal 15 minutes down collects the inclusion body (IBs) through cleaning.With 50mMTris-HCl, pH 8.0 cleans last with purified IBs, and stores in-80 ℃ up to needs with the cell precipitation form.

The IB material is thawed and be dissolved in the 8M carbamide of pH4.0.The optionally stable target protein of this step can be by the solid precipitation of centrifugal removal and the fragment of significant quantity and impurity protein remains are.Because there is single cysteine in these protein, so all subsequent purification programs are to use and contain 1mM DTT and finish as the buffer of Reducing agent.(30ml XK16) catches agarose, and optionally is dissolved in 25mM acetic acid Na (C with 8M carbamide to use SP to circulate fast solubilising protein ₂H ₃O ₂Na), pH 4.0,1mMDTT, 1mM EDTA and 0.2M NaCl eluting.Be to remove the protein precipitation after this step, satisfy before protein refolding, by to 50mM Tris-HCl, pH 8.0 with the pH value of SP eluate, 1mMDTT, and 1mM EDTA dialysis, and be adjusted to 8.0 from 4.0.With through the material of dialysis by directly dilution (10-doubly) to 50mM Tris-HCl pH 8.0,1mM DTT and 1mM EDTA so that final protein concentration is less than 0.1mg/ml, and carry out folding again.To compile through folding again SP direct application of sample to the high-effect agarose of Q (30ml, XK16) on, and bonded albumen is stored in 50mM Tris-HCl with 20 tubing string volumes from 0 to 0.5M NaCl, pH 8.0,1mM DTT, the linear gradient elution among the 1mM EDTA.It is the peak that about 15ms/cm eluting goes out in conductivity that this tomography step produces single.This eluate is compiled and store in-80 ℃.

Protein characteristicization: use following dissecting needle to proteinic purity, homogeneity, endotoxin content and characteristic of biological activityization.

SDS-PAGE: protein (5 μ g typically) is diluted in the SDS-PAGE sample buffer (1% SDS, 30mM Tris-HCl, pH 6.8,4% glycerol, 0.1mg/ml bromophenol blue) that contains or do not contain the 5mM beta-mercaptoethanol.Sample is boiled 5 minutes, and application of sample is to the 4-20% sds page.In the electrophoresis postoperative, gel is dyeed to present protein band so that Kao Maxi is blue.

Endotoxin analysis: use the QCL-1000 LAL test kit (BioWhittaker#50-648U) that quantitatively develops the color,, measure endotoxin concns according to the explanation that manufacturer is used for the micro plate method.

Protein analysis: by with 96-hole form, use the MicroBCA protein analysis reagent test kit (Pierre Si biotechnology (Pierce Biotechnology)) of BSA, measure protein concentration according to the explanation of manufacturer as reference material.

Flagellin ELISA: by the ELISA that carries out with the antibody that is specific to flagellin, check protein integrity and concentration.Under 4 ℃, the serial dilution (starting from 5 μ g/ml) that is dissolved in PBS with each target protein applies and spends the night with 96-hole elisa plate.With the analysis dilution buffer liquid (ADB of plate with 200 μ l/ holes; BD Fa Minggen) under room temperature, blockaded one hour, then in the phosphate-buffered saline solution that contains Tween-20 (PBS-T, 12mM NaPO ₄, 137mM NaCl, 2.7mM KCl, 0.05% Tween 20) and middle the cleaning three times.The many strains of rabbit are resisted-(100 μ l/ holes 1:5000) add in whole holes flagellin antibody, and plate was cultivated under room temperature 1 hour, or spend the night under 4 ℃, clean three times with PBS-T then in the diluent of ADB.With the HRP-labelled goat that is diluted in ADB anti--rabbit igg antibody (Jackson's immunochemistry) adds that (1:5000), and cultivated plate 1 hour in 100 μ l/ holes under room temperature.Plate is cleaned three times with PBS-T.TMB Ultra substrate to be added (Pierre Si), and behind the monitoring color colour generation, on Tecan Farcyte micro plate spectrophotometer, measure A ₄₅₀

The TLR5 biological activity is analyzed: (TLR5 VA) is expressed in the Manassas to the HEK293 cell inherently for ATCC, Cat#CRL-1573, and in the reaction to the TLR5 citation, secretes several soluble factors (comprising IL-8).With cell inoculation (50,000 cells/well) in the micro plate of 96-hole, and the recombinant type test protein added.Next day, collect conditioned medium, be transferred to clean 96-hole micro plate, and freezing down in-20 ℃.After waiting to thaw, in the antibody that uses Anti-Human's class-IL-8 to cooperate to (Pierre Si, Luo Kefu, IL; #M801E and #M802B) sandwich ELISA, exist according to the IL-8 in the explanation analysis condition culture medium of manufacturer.Use micro plate spectrophotometer measurement optical density (OD).

Result and discussion

Protein output and purity: each proteinic ultimate output and endotoxin content degree list as follows:

Protein	SEQ ID NO：	Output (mg)	Endotoxin (EU/ μ g)
				STF2Δ.3’Cys	332	75.0	0.01
The STF2 Δ. hinge Cys	333	117.0	0.004

The flagellin globality: with single cysteine engineered in the STF2 Δ, do not lower its identification, as shown in figure 22 by flagellin-specific antibody.

TLR5 agonist activity in the HEK293IL-8 analysis: with single cysteine engineered in the STF2 Δ, do not lower this proteic TLR agonist activity, as shown in figure 23.Through being exposed to STF2 Δ .3 ' Cys (SEQ ID NO:332) or STF2 Δ. the cell of hinge Cys (SEQ ID NO:333), secretion is with described via being exposed to total length flagellin (STF2, or not have STF2 Δ (SEQ ID NO:313) the institute inductive IL-8 secretion degree of cysteine residues suitable SEQ ID NO:312).

Total length flagellin (STF2, or hinge region SEQ ID NO:312) ,-through the flagellin (STF2 Δ, SEQ ID NO:313) and the proteantigen of disappearance, for example west nile virus envelope protein or influenza A hemagglutinin merge, and obviously increase this immunogenicity through fused antigen.This motion can be used for, and it can be encoded in and make flagellin and institute's interest antigen presentation be single protein, the gene fusion construct in protein or peptide class antigen, but it can't be applied to non--protein or non--peptide class antigen, for example polysaccharide antigen.Because the performance of many antibacterials and tumor cell, but it has the specific polysaccharide structure of antigenicity immunogenicity difference, so but invent out in order to the immunogenicity that increases this class formation and protect the quantitative approach of effect very important.With its chemical conjugation to TLR part (for example flagellin) is an ergastic strategy.

Several chemical connection process are arranged, but wherein the simplest person is coupled to the free state mercapto with antigen, for example is positioned at single, unpaired cysteine residues.Because flagellin is in not containing any cysteine naturally down, so three positions that flagellin is gone in the cysteine residues transformation wherein one: the amino terminal of flagellin (5 ' Cys.STF2 Δ, SEQ ID NO:326 and SEQ ID NO:334), carboxyl terminal (the STF2 Δ .3 ' Cys of flagellin, SEQ ID NO:325 and SEQ ID NO:332) and in the quilt disappearance hinge region of flagellin (the STF2 Δ. hinge Cys, SEQ ID NO:331 and SEQ ID NO:333).All constructs all make in the expression vector pET2Aa that is used for escherichia coli expression.When the Bacillus coli cells purification gets, the STF2 Δ. hinge Cys (SEQ ID NO:333) and STF2 Δ .3 ' Cys (SEQ ID NO:332) protein are possessed whole antigenicities, with the proteinic TLR5 biological activity of not modified STF2 Δ characteristic, therefore determine to import single cysteine residues, expression that can't negative impact STF2 Δ, purification, folding or biological activity again.

Ripe position peptide and the STF2 Δ sheared of influenza virus haemagglutinin. the conjugation of hinge CYS

To represent the peptide of the ripe shearing site of influenza virus haemagglutinin, with the modified flagellin of chemical mode conjugation, STF2 Δ. hinge Cys (SEQ ID NO:333).

Materials and methods

Make the STF2 Δ. hinge Cys protein: as described in previous embodiment, expression and purification get the STF2 Δ. hinge Cys protein (SEQ ID NO:333).

Synthetic H1C1 peptide: sequence NH ₂-NIPSIQSRGLFFAIAGFIE-COOH (SEQ ID NO:337) represents the ripe shearing site (than peace contract, people such as E. (2005)) of influenza virus A/H1N1 hemagglutinin.Universality influenza B vaccine is based on the ripe shearing site (than peace contract, people such as E., J.Virol.79:7380-7388 (2005)) of hemagglutinin predecessor.Design two kinds and have extra cysteine in amino-end, or the extra peptide of lysine to help chamical binding to carrier protein, produce following peptide sequence:

CysH1C1(SEQ ID NO：338)NH ₂-CNIPSIQSRGLFFAIAGFIE-COOH(SEQID NO：338)

LysH1C1(SEQ ID NO：339)NH ₂-KNIPSIQSRGLFFAIAGFIE-COOH(SEQID NO：339)

Described peptide is to receive Spark limited company by peace (Sang Qiongsi CA), uses standard Fmoc chemosynthesis to get, and afterwards they is gone out from the base material cracking with trifluoroacetic acid (TFA), via anti-phase HPLC purification and lyophilization for Anaspec, Inc..

CysH1C1 peptide and STF2 Δ. the conjugation of hinge Cys: with concentration is the STF2 Δ of 4.9mg/mL. hinge Cys protein (SEQ ID NO:333), carry out dialysed overnight in buffer A [1x phosphate-buffering saline solution (PBS), 5mM EDTA, pH7.2].With BM (PEO) ₂(IL), it is a kind of with difunctionality maleimide cross-linking agent for Pierre's Si biotechnology, Luo Kefo, be dissolved among the DMSO (dimethyl sulfoxide), and to add to final concentration is 2.3mM.Treat after cultivating 1 hour under the room temperature, use 5ml Superdex25HiTrap desalination tubing string, the free state cross-linking agent is removed from protein.CysH1C1 peptide (SEQID NO:338) is dissolved among the DMSO, and add to final concentration be 0.32mM through maleimide derivatization STF2 Δ. hinge Cys protein.Treat after cultivating 3 hours under the room temperature, to be the 40mM cessation reaction by DTT (1,4-Dithioerythritol) is added to final concentration.By in through in 1x phosphate-buffering saline solution (PBS), equilibrated Superdex 200 10/300 size exclusion tomography (SEC) tubing string (GE/ Ai Moxun bioscience among the pH 8.0; Skin SIKA tower prestige is carried out fraction on NJ), and protein and protein-peptide conjugates is separated with free peptide.

Protein characteristicization: as described in previous embodiment, to proteinic purity, homogeneity, endotoxin content and characteristic of biological activityization.

Result and discussion

The STF2 Δ. the characterization of hinge Cys:CysH1C1 peptide conjugates: by with the blue painted SDS-PAGE of Kao Maxi, analyze CysH1C1 peptide (SEQ ID NO:338) and STF2A. hinge Cys STF2 Δ. the conjugation of hinge Cys (SEQ ID NO:333).Peptide and protein conjugation produce dual band, and wherein faster mobile band is equivalent to not-through conjugated protein, and more mobile band is equivalent to protein-peptide conjugates.

Because this peptide dissolubility is low, observes vaporific precipitation in the conjugation mixture.For determining whether protein-peptide conjugates precipitates, satisfy mixture centrifugal (16, following 15 minutes of 000xg), and the precipitation and the supernatant that are become are analyzed by SDS-PAGE.Most of peptide-conjugated protein remaines in the supernatant, represents that it still is solubility.

Carry out the STF2 Δ. the size exclusion tomography (SEC) of hinge Cys:CysH1C1 conjugate, so that protein is with residual cross-linking agent and separate without conjugated peptide.The included scope wash-in of tubing string volume that occurs monomer STF2 Δ in prediction is deviate from single main peak, and considerably less material eluting in voidage (Figure 24) is arranged simultaneously, and the expression most protein is a monomer.

The STF2 Δ. the monomer characteristic of hinge Cys:CysH1C1 conjugate is by S200 stream part is carried out the SDS-PAGE analysis confirmation.The going out without the conjugated protein co-elute of same ratio in protein-peptide conjugates and the application of sample sample represents that this conjugate is a monomer.

Through finding the STF2 Δ. the biological activity of hinge Cys:CysH1C1 conjugation mixture with equate BM (PEO) on expression is attached without conjugated STF2A. hinge Cys protein ₂Cross-linking agent and peptide conjugation reaction do not suppress TLR5 stimulating activity (Fig. 8).

Influenza virus haemagglutinin antigen with contain PAM ₃The conjugation of CYS-lipopeptid

It is activated by lipid or lipid-conjugate that several type clock sample receptors (TLR) are arranged.These comprise TLR2/1 (diacyl lipid albumen and GPI-connecting protein matter), TLR2/6 (three acyl group lipoproteins and GPI-connecting protein matter) and TLR4 (liopopolysaccharides).By through binding so far the purposes of the vaccine formed of the antigen of lipoids be tangible: if lipid is the strong activator of innate immunity approach, but itself has immunogenicity.Yet the manufacturing of this type of conjugate is very complicated.The reorganization biosynthesis of lipoprotein in antibacterial is subject to low yield and institute and becomes the purification of protein-lipopeptid fusions quite difficult.The antigenic chemosynthesis of lipopeptid is more direct, but is limited to peptide antigen.At this, we proof intrinsicly is present in the two biosynthetic difficulty with a kind of walking around, and finishes the processing procedure of the antigenic chemosynthesis of lipidization, carries out Pam ₃Cys lipopeptid (a kind of TLR2/6 agonist) and influenza HA 1-1His ₆Chemical conjugation.

Materials and methods

Make HA1-1His ₆ (PR8) Bv (SEQ ID NO:179): the HA1-1His that is prepared into as described herein ₆(PR8) Bv.

Synthetic Pam ₃ CS (K) ₄ GC (SEQ ID NO:335): (Sang Qiongsi, CA), it is synthetic to use standard Fmoc chemistry to carry out general lipopeptid, afterwards they is gone out from the base material cracking with trifluoroacetic acid (TFA), via anti-phase HPLC purification and lyophilization to receive Spark limited company by peace.

Pam ₃ CS (K) ₄ GC (SEQ ID NO:335) and HA1-1His ₆ (PR8) Bv (SEQ ID NO:179) Conjugation: with HA1-1His ₆(PR8) Bv (SEQ ID NO:179) dialyses to buffer A (1x PBS+5mM EDTA, pH 7.2).With Heterobifunctional maleimide/NHS cross-linking agent sulfo group-SMCC (Pierre Si; The Lip river Crane IL) is dissolved among the DMSO, and to add to final concentration be 0.432mM.Treat after cultivating 30min. under the room temperature, protein to be used G-25HiTrap desalination tubing string (GE/ Ai Moxun bioscience; Skin SIKA tower prestige, NJ), desalination is to buffer A.With Triton X-114 (TX-114) (sigma (Sigma); The Saint Louis city, MO) being added to final concentration is 1% (w/v).With Pam ₃CS (K) 4GC is dissolved among the DMSO to 20mg/ml, and adds that to reach final concentration be 0.6mg/ml.Treat after cultivating 3 hours under the room temperature, to place 37 ℃ of baths to reach 10 minutes reaction tube, so that the TX-114 drop forms.Then with sample in 16, under the 000xg centrifugal 10 minutes, to separate cleaning agent and water.After the water sucking-off, with cleaning agent with the buffer A resuspending to the primitive reaction volume.Analyze overall, cleaning agent and contain water sample by SDS-PAGE then.

Protein characteristicization: protein-lipopeptid conjugate is by the SDS-PAGE characterization.Sample (5 μ g typically) is diluted in the SDS-PAGE sample buffer (0.1M Tris, pH 8.0/4% SDS/25% glycerol/0.1M DTT).Sample is boiled 5 minutes, and application of sample is to the 4-20% sds page.In the electrophoresis postoperative, gel is dyeed to present protein band so that Kao Maxi is blue.

Result and discussion

Input HA1-1His ₆(PR8) Bv (SEQ ID NO:179) presents dual band in SDS-PAGE.This is at all HA1-1His that make in baculovirus at present ₆Protein is observed, and it is seemingly because due to the difference of saccharifying.Sulfo group-SMCC derivatization but not with the conjugated HA1-1His of lipopeptid ₆(PR8) Bv (SEQ ID NO:179) still stays aqueous phase when carrying out the TX-114 separable programming, is solubility, the desired characteristic of globular protein.Through conjugation to Pam ₃The HA1-1His of Cys ₆(PR8) Bv (SEQ ID NO:179) presents 3 or 4 bands that have than derivatization input protein high molecular amount, meets it by the lipopeptid covalent modification.In carrying out TX-114 when being separated, higher mw species mainly are partitioned in the cleaning agent mutually, and low species (being equivalent to without modifying protein) are still stayed aqueous phase.This characteristic is isolated from the cleaning agent drop with general observed lipoprotein and conforms to, and definite HA1-1His ₆(PR8) Bv (SEQ ID NO:179) is with Pam ₃CS (K) ₄GC (SEQ ID NO:335) lipopeptid covalent modification.About 60,120 and the higher molecular weight band of 180kDa (its in SMCC-derivatization protein, observing, but in this input, do not occur), be the covalency HA1-1 polymer that forms by cross-linking agent.This multimerization will be by going among the HA1-1 cysteine engineered, and use cysteine-specificity but not lysine-specificity cross-linking agent prevents.This type of countermeasure also can give preferable control lipopeptid and be attached to site on the HA1-1, produces the product of homogenizing more and may promote immunogenicity.

SEQ ID NO:312, the aminoacid sequence of STF2

MAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANRFTANIKG

LTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRL

NEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDSLNVQKAYD

VKDTAVTTKAYANNGTTLDVSGLDDAAIKAATGGTNGTASVTGGAVKFDADNNKYFVTIG

GFTGADAAKNGDYEVNVATDGTVTLAAGATKTTMPAGATTKTEVQELKDTPAVVSADAKN

ALIAGGVDATDANGAELVKMSYTDKNGKTIEGGYALKAGDKYYAADYDEATGAIKAKTTS

YTAADGTTKTAANQLGGVDGKTEVVTIDGKTYNASKAAGHDFKAQPELAEAAAKTTENPL

QKIDAALAQVDALRSDLGAVQNRFNSAITNLGNTVNNLSEARSRIEDSDYATEVSNMSRA

QILQQAGTSVLAQANQVPQNVLSLLR

SEQ ID NO:313, the aminoacid sequence of STF2 Δ

MAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANRFTANIKG

LTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRL

NEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDSLNVH GAPV

DPASPWTENPLQKIDAALAQVDALRSDLGAVQNRFNSAITNLGNTVNNLSEARSRIEDSD

YATEVSNMSRAQILQQAGTSVLAQANQVPQNVLSLLR

SEQ ID NO:314, the nucleotide sequence of STF2 Δ

ATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAA

TCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGC

GCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGT

CTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGC

GCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCT

AACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTG

AACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAG

GACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTG

AAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCATGGAGCGCCGGTG

GATCCTGCTAGCCCATGGACCGAAAACCCGCTGCAGAAAATTGATGCCGCGCTGGCGCAG

GTGGATGCGCTGCGCTCTGATCTGGGTGCGGTACAAAACCGTTTCAACTCTGCTATCACC

AACCTGGGCAATACCGTAAACAATCTGTCTGAAGCGCGTAGCCGTATCGAAGATTCCGAC

TACGCGACCGAAGTTTCCAACATGTCTCGCGCGCAGATTTTGCAGCAGGCCGGTACTTCC

GTTCTGGCGCAGGCTAACCAGGTCCCGCAGAACGTGCTGTCTCTGTTACGT

SEQ ID NO:315, the nucleotide sequence of STF2

ATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAA

TCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGC

GCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGT

CTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGC

GCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCT

AACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTG

AACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAG

GACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTG

AAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCAGAAAGCGTATGAT

GTGAAAGATACAGCAGTAACAACGAAAGCTTATGCCAATAATGGTACTACACTGGATGTA

TCGGGTCTTGATGATGCAGCTATTAAAGCGGCTACGGGTGGTACGAATGGTACGGCTTCT

GTAACCGGTGGTGCGGTTAAATTTGACGCAGATAATAACAAGTACTTTGTTACTATTGGT

GGCTTTACTGGTGCTGATGCCGCCAAAAATGGCGATTATGAAGTTAACGTTGCTACTGAC

GGTACAGTAACCCTTGCGGCTGGCGCAACTAAAACCACAATGCCTGCTGGTGCGACAACT

AAAACAGAAGTACAGGAGTTAAAAGATACACCGGCAGTTGTTTCAGCAGATGCTAAAAAT

GCCTTAATTGCTGGCGGCGTTGACGCTACCGATGCTAATGGCGCTGAGTTGGTCAAAATG

TCTTATACCGATAAAAATGGTAAGACAATTGAAGGCGGTTATGCGCTTAAAGCTGGCGAT

AAGTATTACGCCGCAGATTACGATGAAGCGACAGGAGCAATTAAAGCTAAAACTACAAGT

TATACTGCTGCTGACGGCACTACCAAAACAGCGGCTAACCAACTGGGTGGCGTAGACGGT

AAAACCGAAGTCGTTACTATCGACGGTAAAACCTACAATGCCAGCAAAGCCGCTGGTCAT

GATTTCAAAGCACAACCAGAGCTGGCGGAAGCAGCCGCTAAAACCACCGAAAACCCGCTG

CAGAAAATTGATGCCGCGCTGGCGCAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTA

CAAAACCGTTTCAACTCTGCTATCACCAACCTGGGCAATACCGTAAACAATCTGTCTGAA

GCGCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCG

CAGATTCTGCAGCAGGCCGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTCCCGCAGAAC

GTGCTGTCTCTGTTACGT

SEQ ID NO:316, the nucleotide sequence of STF2.OVA

ATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAA

TCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGC

GCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGT

CTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGC

GCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCT

AACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTG

AACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAG

GACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTG

AAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCAGAAAGCGTATGAT

GTGAAAGATACAGCAGTAACAACGAAAGCTTATGCCAATAATGGTACTACACTGGATGTA

TCGGGTCTTGATGATGCAGCTATTAAAGCGGCTACGGGTGGTACGAATGGTACGGCTTCT

GTAACCGGTGGTGCGGTTAAATTTGACGCAGATAATAACAAGTACTTTGTTACTATTGGT

GGCTTTACTGGTGCTGATGCCGCCAAAAATGGCGATTATGAAGTTAACGTTGCTACTGAC

GGTACAGTAACCCTTGCGGCTGGCGCAACTAAAACCACAATGCCTGCTGGTGCGACAACT

AAAACAGAAGTACAGGAGTTAAAAGATACACCGGCAGTTGTTTCAGCAGATGCTAAAAAT

GCCTTAATTGCTGGCGGCGTTGACGCTACCGATGCTAATGGCGCTGAGTTGGTCAAAATG

TCTTATACCGATAAAAATGGTAAGACAATTGAAGGCGGTTATGCGCTTAAAGCTGGCGAT

AAGTATTACGCCGCAGATTACGATGAAGCGACAGGAGCAATTAAAGCTAAAACCACAAGT

TATACTGCTGCTGACGGCACTACCAAAACAGCGGCTAACCAACTGGGTGGCGTAGACGGT

AAAACCGAAGTCGTTACTATCGACGGTAAAACCTACAATGCCAGCAAAGCCGCTGGTCAT

GATTTCAAAGCACAACCAGAGCTGGCGGAAGCAGCCGCTAAAACCACCGAAAACCCGCTG

CAGAAAATTGATGCCGCGCTGGCGCAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTA

CAAAACCGTTTCAACTCTGCTATCACCAACCTGGGCAATACCGTAAACAATCTGTCTGAA

GCGCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCG

CAGATTTTGCAGCAGGCCGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTCCCGCAGAAC

GTGCTGTCTCTGTTACGTCTCGAGGGCTCCATCGGCGCAGCAAGCATGGAATTTTGTTTT

GATGTATTCAAGGAGCTCAAAGTCCACCATGCCAATGAGAACATCTTCTACTGCCCCATT

GCCATCATGTCAGCTCTAGCCATGGTATACCTGGGTGCAAAAGACAGCACCAGGACACAA

ATAAATAAGGTTGTTCGCTTTGATAAACTTCCAGGATTCGGAGACAGTATTGAAGCTCAG

TGTGGCACATCTGTAAACGTTCACTCTTCACTTAGAGACATCCTCAACCAAATCACCAAA

CCAAATGATGTTTATTCGTTCAGCCTTGCCAGTAGACTTTATGCTGAAGAGAGATACCCA

ATCCTGCCAGAATACTTGCAGTGTGTGAAGGAACTGTATAGAGGAGGCTTGGAACCTATC

AACTTTCAAACAGCTGCAGATCAAGCCAGAGAGCTCATCAATTCCTGGGTAGAAAGTCAG

ACAAATGGAATTATCAGAAATGTCCTTCAGCCAAGCTCCGTGGATTCTCAAACTGCAATG

GTTCTGGTTAATGCCATTGTCTTCAAAGGACTGTGGGAGAAAGCATTTAAGGATGAAGAC

ACACAAGCAATGCCTTTCAGAGTGACTGAGCAAGAAAGCAAACCTGTGCAGATGATGTAC

CAGATTGGTTTATTTAGAGTGGCATCAATGGCTTCTGAGAAAATGAAGATCCTGGAGCTT

CCATTTGCCAGTGGGACAATGAGCATGTTGGTGCTGTTGCCTGATGAAGTCTCAGGCCTT

GAGCAGCTTGAGAGTATAATCAACTTTGAAAAACTGACTGAATGGACCAGTTCTAATGTT

ATGGAAGAGAGGAAGATCAAAGTGTACTTACCTCGCATGAAGATGGAGGAAAAATACAAC

CTCACATCTGTCTTAATGGCTATGGGCATTACTGACGTGTTTAGCTCTTCAGCCAATCTG

TCTGGCATCTCCTCAGCAGAGAGCCTGAAGATATCTCAAGCTGTCCATGCAGCACATGCA

GAAATCAATGAAGCAGGCAGAGAGGTGGTAGGGTCAGCAGAGGCTGGAGTGGATGCTGCA

AGCGTCTCTGAAGAATTTAGGGCTGACCATCCATTCCTCTTCTGTATCAAGCACATCGCA

ACCAACGCCGTTCTCTTCTTTGGCAGATGTGTTTCCCCTTCGAAGCTTGAAGGTAAGCCT

ATCCCTAACCCTCTCCTCGGTCTCGATTCTACGCGTACCGGTCATCATCACCATCACCAT

TGA

SEQ ID NO:317, the nucleotide sequence of STF28BGF-1

CTCGGGAGATCTGCACAAGTAATCAACACTAACAGTCT

SEQ ID NO:318, the nucleotide sequence of STF28MCR-1

CCATGGGCTAGCAGGATCCACCGGCGCTCCCTGCACGTTCA

SEQ ID NO:319, the nucleotide sequence of STF28MCF-2

GGAGCGCCGGTGGATCCTGCTAGCCCATGGACCGAAAACCCG

SEQ ID NO:320, the nucleotide sequence of STF28ECR-2

TCTGCAGAATTCACGTAACAGAGACAGCACGTTCTGCGGGACGTCCCGCAGAACGTGCTG

TCTCTGTTACGTGAATTCTGCAGA

SEQ ID NO:312, the nucleotide sequence of 3 ' forward 1

ACTGAGTGCATATGGCACAAGTAATCAACACTAACAG

SEQ ID NO:322,3 ' reverse 1 nucleotide sequence

GACTGACTGCTCAGCCTATTAGCAGAGCGGCCGCCACTGTGCTGGATATCTAGAG

SEQ ID NO:323, the nucleotide sequence of 5 ' forward 2

AGTCAGGCCATATGTGCGCACAAGTAATCAACACTAACAGTCTG

SEQ ID NO:324,5 ' reverse 2 nucleotide sequence

GACTGACTGCTCAGCCTATTAACGTAACAGAGACAGCACGTTCTGCGGGACCTGGTTAG

SEQ ID NO:325, the nucleotide sequence of STF2 Δ .3 ' Cys

ATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAA

TCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGC

GCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGT

CTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGC

GCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCT

AACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTG

AACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAG

GACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTG

AAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCATGGAGCGCCGGTG

GATCCTGCTAGCCCATGGACCGAAAACCCGCTGCAGAAAATTGATGCCGCGCTGGCGCAG

GTGGATGCGCTGCGCTCTGATCTGGGTGCGGTACAAAACCGTTTCAACTCTGCTATCACC

AACCTGGGCAATACCGTAAACAATCTGTCTGAAGCGCGTAGCCGTATCGAAGATTCCGAC

TACGCGACCGAAGTTTCCAACATGTCTCGCGCGCAGATTTTGCAGCAGGCCGGTACTTCC

GTTCTGGCGCAGGCTAACCAGGTCCCGCAGAACGTGCTGTCTCTGTTACGTGAATTCTCT

AGATATCCAGCACAGTGGCGGCCGCTCTGC

SEQ ID NO:326, the nucleotide sequence of 5 ' Cys.STF2 Δ

ATGTGCGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAAC

AAATCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAAC

AGCGCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAA

GGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAA

GGCGCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCT

GCTAACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGC

CTGAACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCG

CAGGACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGAT

CTGAAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCATGGAGCGCCG

GTGGATCCTGCTAGCCCATGGACCGAAAACCCGCTGCAGAAAATTGATGCCGCGCTGGCG

CAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTACAAAACCGTTTCAACTCTGCTATC

ACCAACCTGGGCAATACCGTAAACAATCTGTCTGAAGCGCGTAGCCGTATCGAAGATTCC

GACTACGCGACCGAAGTTTCCAACATGTCTCGCGCGCAGATTTTGCAGCAGGCCGGTACT

TCCGTTCTGGCGCAGGCTAACCAGGTCCCGCAGAACGTGCTGTCTCTGTTACGT

SEQ ID NO:327, the nucleotide sequence of hinge forward 1

ACTGAGTGCATATGGCACAAGTAATCAACACTAACAG

SEQ ID NO:328, the nucleotide sequence of hinge reverse 3

GGTCCATGGGCAAGCAGGATCCACCGGCGCT

SEQ ID NO:329, the nucleotide sequence of hinge forward 2

AGCGCCGGTGGATCCTGCTTGCCCATGGACC

SEQ ID NO:330, the nucleotide sequence of hinge reverse 4

GACTGACTGCTCAGCCTATTAACGTAACAGAGACAGCACGTTCTGCGGGACCTGGTTAG

SEQ ID NO:331, the STF2 Δ. the nucleotide sequence of hinge Cys

ATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAA

TCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGC

GCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGT

CTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGC

GCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCT

AACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTG

AACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAG

GACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTG

AAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCATGGAGCGCCGGTG

GATCCTGCTTGCCCATGGACCGAAAACCCGCTGCAGAAAATTGATGCCGCGCTGGCGCAG

GTGGATGCGCTGCGCTCTGATCTGGGTGCGGTACAAAACCGTTTCAACTCTGCTATCACC

AACCTGGGCAATACCGTAAACAATCTGTCTGAAGCGCGTAGCCGTATCGAAGATTCCGAC

TACGCGACCGAAGTTTCCAACATGTCTCGCGCGCAGATTTTGCAGCAGGCCGGTACTTCC

GTTCTGGCGCAGGCTAACCAGGTCCCGCAGAACGTGCTGTCTCTGTTACGT

SEQ ID NO:332, the aminoacid sequence of STF2 Δ .3 ' Cys

MAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANRFTANIKG

LTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRL

NEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDSLNVH GAPV

DPASPWTENPLQKIDAALAQVDALRSDLGAVQNRFNSAITNLGNTVNNLSEARSRIEDSD

YATEVSNMSRAQILQQAGTSVLAQANQVPQNVLSLLREFSRYPAQWRPLC

SEQ ID NO:333, the STF2 Δ. the aminoacid sequence of hinge Cys

MAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANRFTANIKG

LTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRL

NEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDSLNVH GAPV

DPACPWTENPLQKIDAALAQVDALRSDLGAVQNRFNSAITNLGNTVNNLSEARSRIEDSD

YATEVSNMSRAQILQQAGTSVLAQANQVPQNVLSLLR

SEQ ID NO:334, the aminoacid sequence of 5 ' Cys.STF2 Δ

MCAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANRFTANIK

GLTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQR

LNEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDSLNVHGAP

VDPASPWTENPLQKIDAALAQVDALRSDLGAVQNRFNSAITNLGNTVNNLSEARSRIEDS

DYATEVSNMSRAQILQQAGTSVLAQANQVPQNVLSLLR

SEQ ID NO：335，

CSKKKKGC

SEQ ID NO：336，

GAPVDPASPW

Embodiment 14 STF2.4xH1C1, a kind of clone and performance that comprises the fused protein of flagellin (TLR5 agonist) and the ripe shearing of influenza A hemagglutinin position peptide

Materials and methods

The construct design: contain the card casket plasmid (STF2.blp, SEQ ID NO:340) that unique BlpI cuts the position for helping clone the gene that merges with flagellin, use then to be positioned at 3 of flagellin gene ' end.This card casket is by silent mutation (5 '-GTGCTGAGCCTGTTACGT-3 ') being directed in the nucleotide 1501 to 1518 of STF2, produces unique BlpI and cut and make in this plasmid card casket pET24/STF2.blp (SEQ ID NO:340) in.Influenza A hypotype H1 (NIPSIQSRGLFGAIAGFIE with coding one, two, three and four copies; The synthetic gene of ripe shearing site fragment (H1C1) SEQ ID NO:341) carries out the codon optimization and is used for escherichia coli expression, and (DNA2.0 Inc. covers Luo Gongyuan, CA) buys from commercial suppliers.Synthetic gene is gone out with the BlpI enzyme action, and via can compatible termination being bonded to, the pET24/STF2.blp that handles with BlpI and bacterial alkaline phosphatase (BAP).These constructs are difference called after STF2.1xH1C1, STF2.2xH1C1, STF2.3xH1C1 and STF2.4xH1C1 (SEQID NO:342; SEQ ID NO:343; SEQ ID NO:344; SEQ ID NO:345).

Equally, make up and to contain the H1C1 sequence, to help the fusion gene of the ring formation of concatemer, called after STF2.4xH1C2 (SEQ ID NO:346) with a pair of cysteine is a flank.Use similar program to produce and comprise influenza A hypotype H5 (RERRRKKRGLFGAIAGFIE; SEQ ID NO:347) construct of segmental four tandem copies of shearing, called after construct STF2.4xH5C1 (SEQ ID NO:348).Therefore represent hypotype H3 (NVPEKQTRGIFGAIAGFIE; SEQ ID NO:349), H2 (NVPQIESRGLFGAIAGFIE; SEQ ID NO:350) and the B-strain, B/HA (PAKLLKERGFFGAIAGFLE; SEQ ID NO:351) total shearing fragment can conform to listed as described above this experiment motion.In case out of the ordinary, be to use constructed plasmid to transform competent escherichia coli TOP10 cell, and make the recombinant that Analysis and Identification is inferred by PCR screening and estriction map.

By the integrity of DNA sequencing proof construct, and use it for the conversion expressive host, BLR3 (DE3) (Nova gold, Santiago, CA; Cat #69053).Transformant is to screen in the flat board that contains kanamycin (50 μ g/mL), tetracycline (5 μ g/mL) and glucose (0.5%).Picking colony and inoculation are gone in the additional LB culture medium with 25 μ g/ml kanamycin, 12.5 μ g/ml tetracyclines and 0.5% glucose of 2ml, and grow overnight.These cultures of five equilibrium are used for, and the fresh culture of inoculation same medium allotment cultivates it up to OD ₆₀₀=0.6, pass through to add 1mM IPTG this moment, and cultivated 3 hours and the induced protein expression down in 37 ℃.Collecting cell and analysing protein expresses.

SDS-PAGE and western blotting: by gel electrophoresis art and immunoblotting assay, measure protein expression and homogeneity.Cell is collected via centrifugal, and in the Laemmli buffer, dissolved.Each lysate of 10 μ l five equilibriums is diluted in, contains or do not contain in the SDS-PAGE sample buffer of 100mM DTT as Reducing agent.Sample is boiled 5 minutes, and carry out electrophoresis (SDS-PAGE) on application of sample to 10% sds page.With gel with the blue R-250 (Bio-Rad of Kao Maxi; Mo Lisi, CA) dyeing is to present protein band.For Western blotting, be that the capable cell lysates of 0.5 μ l/ is carried out electrophoresis, and through electrotransfer to pvdf membrane, blockade with 5% (w/v) milk powder again.With film with anti--flagellin antibody 6H11 (Inotek; Bei Fuli MA) surveys.Treat that (IL) after the detection, (Pu Meijia, horse ground is gloomy, WI) presents protein band with alkali phosphatase colour generation substrate for Pierre Si, Luo Kelan with alkali phosphatase-conjugated secondary antibodies.Filter out to produce and have correct molecular weight, and with the pure strain of antibacterial of the protein band of suitable antibody response, be used to make protein for being used for bioanalysis.

Result and discussion

The cloning and expression of shearing site construct: the ripe shearing site of hemagglutinin predecessor, between strains of influenza viruses A and B, have suitable conservative, be used to research and develop effective antagonism, the target thing of the general vaccine of most of influenza virus epidemic strains and therefore can be.Produce a series of coding TLR5 parts and the plasmid of shearing segmental fusion rotein, and in e. coli strains BLR3 (DE3), express.Such as by the blue dyeing of the Kao Maxi of SDS-PAGE gel analysis, and by the immunoblotting assay authenticator, the e. coli strains that has construct STF2.1xH1C1, STF2.2xH1C1, STF2.3xH1C1 and STF2.4xH1C1 presents and corresponds respectively to the molecular weight 55,58,60 be scheduled to and 62 band.Equally, the BLR of expression construct STF2.4xH1C2 and STF2.4xH5C1 (DE3) bacterial strain presents and has the fused protein that obvious molecular weight is respectively 62Kda and 66Kda.These data point out, flagellin and HA shear that segmental fusion rotein is abundant to be expressed in escherichia coli.

STF2.4xH1C1, a kind of flagellin (TLR5 agonist) and ripe performance and purification of shearing the fused protein of position peptide of influenza A hemagglutinin of comprising

Materials and methods

Bacterial cell growth and cytolysis: STF2.4xH1C1 (SEQ ID NO:345) construct is expressed among the e. coli host bacteria strain BLR (DE3).Bacterial strain is recovered from the glycerol reserve, and grow in shake in the bottle to final volume be 12L.Cell is grown in the LB culture medium that contains 25 μ g/ml kanamycin/12.5 μ g/ml tetracycline/0.5% glucoses to OD ₆₀₀=0.6, and cultivate down in 37 ℃ with 1mM IPTG and to induce in 3 hours.Cell is collected by centrifugal (7000rpm x 7 minutes in Sorvall RC5C centrifuge), and resuspending is in 20mM Tris-HCl, in pH 8.0,1 μ g/ml DNAseI, 1mM PMSF, protease inhibitor cocktail and the 1mg/ml lysozyme.Then with cell in 15, under the 000psi twice by little liquefier (micro liquid is learned (Microfluidics); Niu Dun, the dissolving that MA) makes.With lysate in Beckman Optima L supercentrifuge (Beckman colter; Fu Ledun, CA) in, in 45, under the 000g centrifugal one hour so that solubility is partly separated with insolubility.

The purification of STF2.4xH1C1 (SEQ ID NO:345): after centrifugal, supernatant liquid stream part is collected, and by Q agarose tubing string (the GE/ Ai Moxun bioscience that circulates fast; Skin SIKA tower prestige, NJ).With since then circulation stream part of step replenish with Triton X-100 (sigma; The Saint Louis city, MO) to final concentration be 1% (w/v), and by identical Q agarose tubing string.Collect circulation stream part once more, and to replenish with carbamide to final concentration be 8M, and citric acid to final concentration is 20mM.After pH value being adjusted to 3.5, solution is passed through through the 20mM citric acid pH 3.5 equilibrated sucrose S tubing string (GE/ Ai Moxun bioscience with dense HCl; Skin SIKA tower prestige, NJ).

Then tubing string is washed with the level pad that 10 tubing string volumes replenish with 1% (w/v) Triton X-100, to remove endotoxin.Then in 5-tubing string volume 0 to 1M NaCl eluting in the linear gradient of level pad.Then STF2.4xH1C1 is carried out in the buffer [0.1M Tris-HCl, pH 8.0,0.1M NaC, 1% (w/v) glycerol] folding to folding by rapid dilution again again.To use pressurization ultra-filtration stirring pool (Mi Libo through folding protein; The Baily card MA) concentrates, and in Superdex 200 sizes-eliminating tubing string (GE/ Ai Moxun bioscience; Skin SIKA tower prestige NJ) is carried out fraction.

SDS-PAGE and western blotting: measure protein homogeneity by SDS-PAGE, and assessment purity.The sample out of the ordinary of 5 μ g five equilibriums is diluted in, contains or do not contain in the SDS-PAGE sample buffer of 100mM DTT as Reducing agent.Sample is boiled 5 minutes, and application of sample to 10% sds page (LifeGels; The blue Si Senlin of method, Xin Nanweiersi, AUS) on, and carry out the electrophoresis art.With gel with the blue R-250 (Bio-Rad of Kao Maxi; Hull Ke Lishi, CA) dyeing is to present protein band.For Western blotting, be that the capable overall protein matter of 0.5 μ l/ is carried out electrophoresis as previously mentioned, through electricity-be transferred on the pvdf membrane, and blockade, then afterwards with anti--flagellin antibody (Inotek with 5% (w/v) milk powder; Bei Fuli MA), or derives from the serum that causes the mice of exempting from through synthesis type, lipid H1C1 peptide and surveys.Treat with alkali phosphatase-conjugated secondary antibodies (Pierre Si; The Lip river Crane, IL) after the detection, (Pu Meijia, horse ground is gloomy, WI) presents protein band with alkali phosphatase colour generation substrate.

Protein analysis: use Micro BCA (two cinchoninic acids) to analyze (Pierre Si; Luo Kefu IL) with the micro plate form, uses bovine serum albumin as reference material, according to the explanation of manufacturer, measures overall protein matter concentration.

Endotoxin analysis: use the QCL-1000 LAL test kit (Cambrex that quantitatively develops the color; E. the endotoxin degree not NJ), according to the explanation that manufacturer is used for the micro plate method, is measured in the Luther.

The TLR5 biological activity is analyzed: the HEK293 cell is expressed TLR5 inherently, and in the reaction to the TLR5 citation, secretes several soluble factors (comprising IL-8).With cell inoculation (50,000 cells/well) in the micro plate of 96-hole, and the STF2.4xH1C1 test protein added.Next day, collect conditioned medium, be transferred to clean 96-hole micro plate, and freezing down in-20 ℃.After waiting to thaw, in the antibody that uses Anti-Human's class-IL-8 to cooperate to (Pierre Si, Luo Kelan, IL; #M801E and #M802B) sandwich ELISA, according to the existence of IL-8 in the explanation analysis condition culture medium of manufacturer.Use micro plate spectrophotometer (FARCyte, GE/ Ai Moxun; Skin SIKA tower prestige NJ) is measured optical density (OD).

Result and discussion

Purified and folding again STF2.4xH1C1 protein (SEQ ID NO:345) is in fact assembled and is the most of protein (Figure 26) of fraction in the voidage of Superdex S200 tubing string through finding.This protein also has the poor activity of LTR5 in vitro, its EC ₅₀Value is approximately higher than standard flagellum fusion rotein STF2.OVA two powers greatly.Purified protein can with derive from the seroreaction that causes the mice of exempting from through synthesis type, lipid H1C1 peptide.

STF2.1xH1C1, a kind of flagellin (TLR5 agonist) and ripe performance and purification of shearing the fused protein of position peptide of influenza A hemagglutinin of comprising

Materials and methods

Bacterial cell growth and cytolysis: STF2.1xH1C1 (SEQ ID NO:355) construct is expressed among the e. coli host bacteria strain BLR (DE3).Bacterial strain is recovered from the glycerol reserve, and grow in shake in the bottle to final volume be 12L.Cell is grown in the LB culture medium that contains 25 μ g/ml kanamycin/12.5 μ g/ml tetracycline/0.5% glucoses to OD ₆₀₀=0.6, and cultivate down in 37 ℃ with 1mM IPTG and to induce in 3 hours.Cell is collected by centrifugal (7000rpm x 7 minutes in Sorvall RC5C centrifuge), and resuspending is in 20mM Tris-HCl, in pH8.0,1 μ g/ml DNAseI, 1mM PMSF, protease inhibitor cocktail and the 1mg/ml lysozyme.Then with cell in 15, under the 000psi twice by little liquefier (micro liquid is learned; Niu Dun, the dissolving that MA) makes.With lysate in Beckman Optima L supercentrifuge (Beckman colter; Fu Ledun, CA) in, in 45, under the 000g centrifugal one hour so that solubility is partly separated with insolubility.

The purification of STF2.1xH1C1 (SEQ ID NO:342): after centrifugal, supernatant liquid stream part is collected, and by Q agarose tubing string (the GE/ Ai Moxun bioscience that circulates fast; Skin SIKA tower prestige, NJ).With since then circulation stream part of step replenish with Triton X-100 (sigma; The Saint Louis city, MO) to final concentration be 1% (w/v), and by identical Q agarose tubing string.Collect circulation stream part once more, and to replenish with carbamide to final concentration be 8M, and citric acid to final concentration is 20mM.After pH value being adjusted to 3.5, solution is passed through through the 20mM citric acid the equilibrated sucrose S of pH3.5 tubing string (GE/ Ai Moxun bioscience with dense HCl; Skin SIKA tower prestige, NJ).

Then tubing string is washed with the level pad that 10 tubing string volumes replenish with 1% (w/v) Triton X-100, to remove endotoxin.Then in 5-tubing string volume 0 to 1M NaCl eluting in the linear gradient of level pad.Then STF2.1xH1C1 is carried out in the buffer [0.1M Tris-HCl, pH8.0/0.1M NaCl/1% (w/v) glycerol] folding to folding by rapid dilution again again.To use pressurization ultra-filtration stirring pool (Mi Libo through folding protein; The Baily card MA) concentrates, and in Superdex 200 sizes-eliminating tubing string (GE/ Ai Moxun bioscience; Skin SIKA tower prestige NJ) is carried out fraction.

SDS-PAGE and western blotting: measure protein homogeneity by SDS-PAGE, and assessment purity.The sample out of the ordinary of 5 μ g five equilibriums is diluted in, contains or do not contain in the SDS-PAGE sample buffer of 100mM DTT as Reducing agent.Sample is boiled 5 minutes, and application of sample to 10% sds page (LifeGels; The blue Si Senlin of method, Xin Nanweiersi, AUS) on, and carry out the electrophoresis art.With gel with the blue R-250 (Bio-Rad of Kao Maxi; Hull Ke Lishi, CA) dyeing is to present protein band.For Western blotting, be that the capable overall protein matter of 0.5 μ l/ is carried out electrophoresis as previously mentioned, through electricity-be transferred on the pvdf membrane, and blockade, then afterwards with anti--flagellin antibody (Inotek with 5% (w/v) milk powder; Bei Fuli, MA), the Pam3Cys.H1C1 peptide that maybe must hang oneself (SEQ ID NO:358) causes the serum of the mice of exempting from and surveys.Treat with alkali phosphatase-conjugated secondary antibodies (Pierre Si; The Lip river Crane, IL) after the detection, (Pu Meijia, horse ground is gloomy, WI) presents protein band with alkali phosphatase colour generation substrate.

Result and discussion

Purified and folding again STF2.1xH1C1 protein (SEQ ID NO:342) is through finding that when differentiating with eluting stream part of Superdex 200 gel filtration tubing strings, it is a monomer.Most of protein is present in through discovery, and eluting goes out being mingled with in the volume of main peak at about 14mls place.This conforms to closely with purified, the known eluting collection of illustrative plates of monomer flagellin on this tubing string.In voidage (the known 7mls that is approximately) for this tubing string, do not find that almost the protein eluting goes out, prove that in fact aggregate-free exists.

PAM3CYS.H1C1, a kind of PAM3CYS (TLR2 agonist) and ripe immunogenicity and effect of shearing the fused protein of position peptide of influenza A hemagglutinin of comprising

Materials and methods

The peptide design is with synthetic: Pam3 (three-palmityl) is the native ligand of clock sample receptor 2 (TLR2), and it expresses the lipid motif that is bacterial lipoprotein (BLP, SEQ ID NO:357) naturally.Pam3 can by chemosynthesis make and conjugation to such as giant molecules such as protein, maybe can use the standard peptide synthetic chemistry to be coupled to the N-end of synthetic peptide.This strategy is usually directed to the synthetic of institute's interest peptide, comprises that the cysteine (Pam3Cys) that will modify through Pam3-is coupled to amino terminal, and produces the lipopeptid of institute's interest.This motion is to be used for synthetic fat materialization HA to shear fragment peptide, Pam3Cys.H1C1 (Pam3Cys-SLWSEENIPSIQSRGLFGAIAGFIEE, SEQ ID NO:358).

Mice is exempted from causing: use the 6-8 female BALB/c mouse in age in week (Jackson's laboratory, Ba Ergang, ME).With 10 every group of mice group, and near accepting groin in the 0th and 14 day subcutaneous (s.c) cause exempt from as follows:

1) PBS (phosphate-buffered saline solution)

2) the H1C1 native peptides of 20 μ g (SEQ ID NO:358) is in saline solution buffer (10mM histidine, 10mM Tris, 75mM NaCl, 5% (vol/vol) sucrose, 0.02% (w/v) gathers sorbose acid esters-80,0.1mM EDTA, 0.5% (v/v) ethanol, pH 7.2)

3) the Pam3Cys.H1C1 peptide of 20 μ g (SEQ ID NO:358) is in the saline solution buffer

Extra one group of five mice is accepted to excite with 8x10 through inferior the causing death that measuring gets ¹Infectious dosage (EID) PR/8/34 of egg, and make its recovery〉21 days.Then with these animals during exciting research (referring to following), use as immune restoration phase positive controls.With mice (the short liter) blood sampling in the 10th day (elementary) and 21 days, and with serum by condensing and centrifugal clarificationization, and store under-20 ℃.

Serum antibody is measured: measure H1C1-specific IgG concentration by ELISA.(Costar (Cat#9018) is healthy and free from worry, NY) under 4 ℃, is dissolved in PBS (5 μ g/ml) with 100 μ l/ hole H1C1 peptides (SEQ ID NO:358) and applies and spend the night with 96-hole elisa plate.With the analysis dilution buffer liquid (ADB of plate with 200 μ l/ holes; BD Fa Minggen, (Cat#:555213) (famous scenic spot tooth brother CA) blockaded under room temperature one hour.Plate is cleaned three times in PBS+0.05% (v/v) Tween 20 (PBS-T).Serum is added (100 μ l/ hole) in the diluent of ADB, and plate cultivation under 4 ℃ is spent the night.Plate is cleaned three times with PBS-T.The HRP-labelled goat that is diluted in ADB is resisted-mouse IgG antibody (Jackson's immunochemistry; Xi Geluofu, PA (Cat#:115-035-146)) add (100 μ l/ hole), and plate was cultivated under room temperature 1 hour.Plate is cleaned three times with PBS-T.(IL), and behind the monitoring color colour generation, (Dole breathes out TMB Ultra substrate to be added, NC) measures A on the micro plate spectrophotometer in Tecan Farcyte for Pierre Si (Cat 34028), Luo Kefu ₄₅₀

Influenza virus excites miceBe to analyze effect, satisfy foregoing, threw by intranasal in the 28th day and give LD through immune mouse ₉₀(making the lethal dosage of 90% mice) (8x10 ³EID) influenza A separated strain PR/8/34 and exciting.Every day is investigated the survival of animal in the back, body weight runs off and clinical sign reaches 21 days in exciting.It is that meansigma methods with every group of animal out of the ordinary ((body weight every day (g)/28th day original body weight (g)) x100) is that basic calculation gets that the % body weight runs off.The appointment of clinical scores is as follows: 4 points=health, and 3 points=minimizing combing, 2 points=health is active to be lowered, and 1 point=dying.(experimental result that clinical scores and body weight run off reflects based on the result of surviving animals in assessed natural law).

Result and discussion

Exempt from inducing of back H1C1-specific antibody reaction in causing with Pam3Cys.H1C1: mice with natural H1C1 peptide (SEQ ID NO:358), and is caused and exempts from by adding identical peptide that amino terminal Pam3Cys residue modifies, peptide and TLR2 part binding can be increased its immunogenic theory with test.Immunogenicity is by measuring in exempting from the mice serum through causing, and resists the antibody degree of natural H1C1 (SEQ ID NO:358) and measures.The result clearly illustrates, causes the mice of exempting from through the Pam3Cys-modified peptides and produces, and it is higher to cause the mice of exempting from through native peptides (SEQ ID NO:358), and antagonism causes the antibody titer (Figure 27) of exempting from the peptide backbone sequence.

The protective effect that excites in the influenza that causes death with avoiding after the Pam3Cys.H1C1 immunity: the result of Figure 27 proves that mice causes with Pam3Cys.H1C1 and exempts to produce the antibody response that can discern natural H1C1 peptide.Be the assessment effect, satisfied in the 28th day and give LD through the intranasal throwing ₉₀(8 x 10 ³EID) PR/8/34 virus and exciting.Every day is investigated the survival of mice in the back, body weight runs off and clinical sign reaches 21 days in exciting.As shown in figure 28, promptly dead between 6 to 10 days after PBS-causes the mice of exempting from and exciting, and convalescent period mice in 21-days observation periods after, still survived.Cause the mice of exempting from through natural H1C1 (SEQ ID NO:358) after the infection before 6, with the speed death similar, though after 21-days observation periods, in 10 mices 2 still survivals were arranged to the PBS control group mice.On the contrary, be similar to the convalescent period mice, all cause mice all still survival after 21-days observation periods of exempting from through Pam3Cys.H1C1.Therefore, though the H1C1 effective immunogenicity of tool (Figure 27) is not separately also had a protectiveness (Figure 28), have immunogenicity and protectiveness concurrently with the identical sequence of TLR2 ligand coupling.

SEQ ID NO：340 STF2.blp

ATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAA

TCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGC

GCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGT

CTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGC

GCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCT

AACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTG

AACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAG

GACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTG

AAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCAGAAAGCGTATGAT

GTGAAAGATACAGCAGTAACAACGAAAGCTTATGCCAATAATGGTACTACACTGGATGTA

TCGGGTCTTGATGATGCAGCTATTAAAGCGGCTACGGGTGGTACGAATGGTACGGCTTCT

GTAACCGGTGGTGCGGTTAAATTTGACGCAGATAATAACAAGTACTTTGTTACTATTGGT

GGCTTTACTGGTGCTGATGCCGCCAAAAATGGCGATTATGAAGTTAACGTTGCTACTGAC

GGTACAGTAACCCTTGCGGCTGGCGCAACTAAAACCACAATGCCTGCTGGTGCGACAACT

AAAACAGAAGTACAGGAGTTAAAAGATACACCGGCAGTTGTTTCAGCAGATGCTAAAAAT

GCCTTAATTGCTGGCGGCGTTGACGCTACCGATGCTAATGGCGCTGAGTTGGTCAAAATG

TCTTATACCGATAAAAATGGTAAGACAATTGAAGGCGGTTATGCGCTTAAAGCTGGCGAT

AAGTATTACGCCGCAGATTACGATGAAGCGACAGGAGCAATTAAAGCTAAAACTACAAGT

TATACTGCTGCTGACGGCACTACCAAAACAGCGGCTAACCAACTGGGTGGCGTAGACGGT

AAAACCGAAGTCGTTACTATCGACGGTAAAACCTACAATGCCAGCAAAGCCGCTGGTCAT

GATTTCAAAGCACAACCAGAGCTGGCGGAAGCAGCCGCTAAAACCACCGAAAACCCGCTG

CAGAAAATTGATGCCGCGCTGGCGCAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTA

CAAAACCGTTTCAACTCTGCTATCACCAACCTGGGCAATACCGTAAACAATCTGTCTGAA

GCGCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCG

CAGATTCTGCAGCAGGCCGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTCCCGCAGAAC

GTGCTGAGCCTGTTACGT

The ripe fragment (H1N1) of shearing of SEQ ID NO:341 HA

NIPSIQSRGLFGAIAGFIE

SEQ ID NO：342 STF2.1xH1C1

MAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANRFTANIKG

LTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRL

NEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDSLNVQKAYD

VKDTAVTTKAYANNGTTLDVSGLDDAAIKAATGGTNGTASVTGGAVKFDADNNKYFVTIG

GFTGADAAKNGDYEVNVATDGTVTLAAGATKTTMPAGATTKTEVQELKDTPAVVSADAKN

ALIAGGVDATDANGAELVKMSYTDKNGKTIEGGYALKAGDKYYAADYDEATGAIKAKTTS

YTAADGTTKTAANQLGGVDGKTEVVTIDGKTYNASKAAGHDFKAQPELAEAAAKTTENPL

QKIDAALAQVDALRSDLGAVQNRFNSAITNLGNTVNNLSEARSRIEDSDYATEVSNMSRA

QILQQAGTSVLAQANQVPQNVLSLLAMEWENIPSIQSRGLFGAIAGFIE

SEQ ID NO：343 STF2.2xH1C1

MAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANRFTANIKG

LTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRL

NEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDSLNVQKAYD

VKDTAVTTKAYANNGTTLDVSGLDDAAIKAATGGTNGTASVTGGAVKFDADNNKYFVTIG

GFTGADAAKNGDYEVNVATDGTVTLAAGATKTTMPAGATTKTEVQELKDTPAVVSADAKN

ALIAGGVDATDANGAELVKMSYTDKNGKTIEGGYALKAGDKYYAADYDEATGAIKAKTTS

YTAADGTTKTAANQLGGVDGKTEVVTIDGKTYNASKAAGHDFKAQPELAEAAAKTTENPL

QKIDAALAQVDALRSDLGAVQNRFNSAITNLGNTVNNLSEARSRIEDSDYATEVSNMSRA

QILQQAGTSVLAQANQVPQNVLSLLAMEWENIPSIQSRGLFGAIAGFIEEWENIPSIQSR

GLFGAIAGFIE

SEQ ID NO：344 STF2.3xH1C1

MAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANRFTANIKG

LTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRL

NEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDSLNVQKAYD

VKDTAVTTKAYANNGTTLDVSGLDDAAIKAATGGTNGTASVTGGAVKFDADNNKYFVTIG

GFTGADAAKNGDYEVNVATDGTVTLAAGATKTTMPAGATTKTEVQELKDTPAVVSADAKN

ALIAGGVDATDANGAELVKMSYTDKNGKTIEGGYALKAGDKYYAADYDEATGAIKAKTTS

YTAADGTTKTAANQLGGVDGKTEVVTIDGKTYNASKAAGHDFKAQPELAEAAAKTTENPL

QKIDAALAQVDALRSDLGAVQNRFNSAITNLGNTVNNLSEARSRIEDSDYATEVSNMSRA

QILQQAGTSVLAQANQVPQNVLSLLAMEWENIPSIQSRGLFGAIAGFIEEWENIPSIQSR

GLFGAIAGFIEEWENIPSIQSRGLFGAIAGFIE

SEQ ID NO：345 STF2.4xH1C1

MAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANRFTANIKG

LTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRL

NEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDSLNVQKAYD

VKDTAVTTKAYANNGTTLDVSGLDDAAIKAATGGTNGTASVTGGAVKFDADNNKYFVTIG

GFTGADAAKNGDYEVNVATDGTVTLAAGATKTTMPAGATTKTEVQELKDTPAVVSADAKN

ALIAGGVDATDANGAELVKMSYTDKNGKTIEGGYALKAGDKYYAADYDEATGAIKAKTTS

YTAADGTTKTAANQLGGVDGKTEVVTIDGKTYNASKAAGHDFKAQPELAEAAAKTTENPL

QKIDAALAQVDALRSDLGAVQNRFNSAITNLGNTVNNLSEARSRIEDSDYATEVSNMSRA

QILQQAGTSVLAQANQVPQNVLSLLAMEWENIPSIQSRGLFGAIAGFIEEWENIPSIQSR

GLFGAIAGFIEEWENIPSIQSRGLFGAIAGFIEEWENIPSIQSRGLFGAIAGFIE

SEQ ID NO：346 STF2.4xH1C2

MAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANRFTANIKG

LTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRL

NEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDSLNVQKAYD

VKDTAVTTKAYANNGTTLDVSGLDDAAIKAATGGTNGTASVTGGAVKFDADNNKYFVTIG

GFTGADAAKNGDYEVNVATDGTVTLAAGATKTTMPAGATTKTEVQELKDTPAVVSADAKN

ALIAGGVDATDANGAELVKMSYTDKNGKTIEGGYALKAGDKYYAADYDEATGAIKAKTTS

YTAADGTTKTAANQLGGVDGKTEVVTIDGKTYNASKAAGHDFKAQPELAEAAAKTTENPL

QKIDAALAQVDALRSDLGAVQNRFNSAITNLGNTVNNLSEARSRIEDSDYATEVSNMSRA

QILQQAGTSVLAQANQVPQNVLSLLAGCGSEWENIPSIQSRGLFGAIAGFIEEWENIPSI

QSRGLFGAIAGFIEEWENIPSIQSRGLFGAIAGFIEEWENIPSIQSRGLFGAIAGFIESG

C

The ripe fragment (H5N1) of shearing of SEQ ID NO:347 HA

RERRRKKRGLFGAIAGFIE

SEQ ID NO：348 STF2.4xH5C1

MAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANRFTANIKG

LTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRL

NEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDSLNVQKAYD

VKDTAVTTKAYANNGTTLDVSGLDDAAIKAATGGTNGTASVTGGAVKFDADNNKYFVTIG

GFTGADAAKNGDYEVNVATDGTVTLAAGATKTTMPAGATTKTEVQELKDTPAVVSADAKN

ALIAGGVDATDANGAELVKMSYTDKNGKTIEGGYALKAGDKYYAADYDEATGAIKAKTTS

YTAADGTTKTAANQLGGVDGKTEVVTIDGKTYNASKAAGHDFKAQPELAEAAAKTTENPL

QKIDAALAQVDALRSDLGAVQNRFNSAITNLGNTVNNLSEARSRIEDSDYATEVSNMSRA

QILQQAGTSVLAQANQVPQNVLSLLAEWERERRRKKRGLFGAIAGFIEEWERERRRKKRG

LFGAIAGFIEEWERERRRKKRGLFGAIAGFIEEWERERRRKKRGLFGAIAGFIE

The ripe fragment (H3N2) of shearing of SEQ ID NO:349 HA

NVPEKQTRGIFGAIAGFIE

The ripe fragment (H2N1/H2N2/H2N3/H2N5/H2N8/H2N9) of shearing of SEQ ID NO:350 HA

NVPQIESRGLFGAIAGFIE

The ripe fragment (B strain) of shearing of SEQ ID NO:351 HA

PAKLLKERGFFGAIAGFLE

SEQ ID NO：352 STF2.4xH1C1

ATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAA

TCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGC

GCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGT

CTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGC

GCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCT

AACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTG

AACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAG

GACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTG

AAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCAGAAAGCGTATGAT

GTGAAAGATACAGCAGTAACAACGAAAGCTTATGCCAATAATGGTACTACACTGGATGTA

TCGGGTCTTGATGATGCAGCTATTAAAGCGGCTACGGGTGGTACGAATGGTACGGCTTCT

GTAACCGGTGGTGCGGTTAAATTTGACGCAGATAATAACAAGTACTTTGTTACTATTGGT

GGCTTTACTGGTGCTGATGCCGCCAAAAATGGCGATTATGAAGTTAACGTTGCTACTGAC

GGTACAGTAACCCTTGCGGCTGGCGCAACTAAAACCACAATGCCTGCTGGTGCGACAACT

AAAACAGAAGTACAGGAGTTAAAAGATACACCGGCAGTTGTTTCAGCAGATGCTAAAAAT

GCCTTAATTGCTGGCGGCGTTGACGCTACCGATGCTAATGGCGCTGAGTTGGTCAAAATG

TCTTATACCGATAAAAATGGTAAGACAATTGAAGGCGGTTATGCGCTTAAAGCTGGCGAT

AAGTATTACGCCGCAGATTACGATGAAGCGACAGGAGCAATTAAAGCTAAAACTACAAGT

TATACTGCTGCTGACGGCACTACCAAAACAGCGGCTAACCAACTGGGTGGCGTAGACGGT

AAAACCGAAGTCGTTACTATCGACGGTAAAACCTACAATGCCAGCAAAGCCGCTGGTCAT

GATTTCAAAGCACAACCAGAGCTGGCGGAAGCAGCCGCTAAAACCACCGAAAACCCGCTG

CAGAAAATTGATGCCGCGCTGGCGCAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTA

CAAAACCGTTTCAACTCTGCTATCACCAACCTGGGCAATACCGTAAACAATCTGTCTGAA

GCGCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCG

CAGATTCTGCAGCAGGCCGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTCCCGCAGAAC

GTGCTGTCTCTGTTAGCGATGGAATGGGAGAACATCCCTAGCATCCAATCTCGCGGCCTG

TTTGGCGCTATCGCGGGCTTTATCGAAGAATGGGAGAACATCCCGAGCATCCAATCTCGC

GGTCTGTTTGGTGCGATCGCTGGTTTCATCGAGGAGTGGGAGAACATTCCTAGCATTCAA

AGCCGTGGCCTGTTCGGCGCTATTGCAGGTTTTATTGAAGAATGGGAAAATATCCCGTCT

ATCCAATCCCGCGGTCTGTTCGGCGCGATCGCAGGTTTCATTGAATAATAAGCTAAGC

SEQ ID NO：353 STF2.3xH1C1

ATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAA

TCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGC

GCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGT

CTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGC

GCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCT

AACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTG

AACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAG

GACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTG

AAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCAGAAAGCGTATGAT

GTGAAAGATACAGCAGTAACAACGAAAGCTTATGCCAATAATGGTACTACACTGGATGTA

TCGGGTCTTGATGATGCAGCTATTAAAGCGGCTACGGGTGGTACGAATGGTACGGCTTCT

GTAACCGGTGGTGCGGTTAAATTTGACGCAGATAATAACAAGTACTTTGTTACTATTGGT

GGCTTTACTGGTGCTGATGCCGCCAAAAATGGCGATTATGAAGTTAACGTTGCTACTGAC

GGTACAGTAACCCTTGCGGCTGGCGCAACTAAAACCACAATGCCTGCTGGTGCGACAACT

AAAACAGAAGTACAGGAGTTAAAAGATACACCGGCAGTTGTTTCAGCAGATGCTAAAAAT

GCCTTAATTGCTGGCGGCGTTGACGCTACCGATGCTAATGGCGCTGAGTTGGTCAAAATG

TCTTATACCGATAAAAATGGTAAGACAATTGAAGGCGGTTATGCGCTTAAAGCTGGCGAT

AAGTATTACGCCGCAGATTACGATGAAGCGACAGGAGCAATTAAAGCTAAAACTACAAGT

TATACTGCTGCTGACGGCACTACCAAAACAGCGGCTAACCAACTGGGTGGCGTAGACGGT

AAAACCGAAGTCGTTACTATCGACGGTAAAACCTACAATGCCAGCAAAGCCGCTGGTCAT

GATTTCAAAGCACAACCAGAGCTGGCGGAAGCAGCCGCTAAAACCACCGAAAACCCGCTG

CAGAAAATTGATGCCGCGCTGGCGCAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTA

CAAAACCGTTTCAACTCTGCTATCACCAACCTGGGCAATACCGTAAACAATCTGTCTGAA

GCGCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCG

CAGATTCTGCAGCAGGCCGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTCCCGCAGAAC

GTGCTGAGCCTGTTAGCGATGGAATGGGAAAATATCCCTAGCATCCAATCTCGCGGTCTG

TTCGGTGCTATTGCTGGCTTCATCGAGGAATGGGAGAACATCCCATCTATTCAGTCTCGC

GGCCTGTTTGGTGCGATCGCGGGTTTTATTGAGGAATGGGAAAACATTCCAAGCATTCAG

TCACGTGGTCTTTTCGGCGCCATCGCTGGTTTTATCGAATGATAAGCTTAGCCCAAGG

SEQ ID NO：354 STF2.2xH1C1

ATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAA

TCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGC

GCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGT

CTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGC

GCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCT

AACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTG

AACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAG

GACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTG

AAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCAGAAAGCGTATGAT

GTGAAAGATACAGCAGTAACAACGAAAGCTTATGCCAATAATGGTACTACACTGGATGTA

TCGGGTCTTGATGATGCAGCTATTAAAGCGGCTACGGGTGGTACGAATGGTACGGCTTCT

GTAACCGGTGGTGCGGTTAAATTTGACGCAGATAATAACAAGTACTTTGTTACTATTGGT

GGCTTTACTGGTGCTGATGCCGCCAAAAATGGCGATTATGAAGTTAACGTTGCTACTGAC

GGTACAGTAACCCTTGCGGCTGGCGCAACTAAAACCACAATGCCTGCTGGTGCGACAACT

AAAACAGAAGTACAGGAGTTAAAAGATACACCGGCAGTTGTTTCAGCAGATGCTAAAAAT

GCCTTAATTGCTGGCGGCGTTGACGCTACCGATGCTAATGGCGCTGAGTTGGTCAAAATG

TCTTATACCGATAAAAATGGTAAGACAATTGAAGGCGGTTATGCGCTTAAAGCTGGCGAT

AAGTATTACGCCGCAGATTACGATGAAGCGACAGGAGCAATTAAAGCTAAAACTACAAGT

TATACTGCTGCTGACGGCACTACCAAAACAGCGGCTAACCAACTGGGTGGCGTAGACGGT

AAAACCGAAGTCGTTACTATCGACGGTAAAACCTACAATGCCAGCAAAGCCGCTGGTCAT

GATTTCAAAGCACAACCAGAGCTGGCGGAAGCAGCCGCTAAAACCACCGAAAACCCGCTG

CAGAAAATTGATGCCGCGCTGGCGCAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTA

CAAAACCGTTTCAACTCTGCTATCACCAACCTGGGCAATACCGTAAACAATCTGTCTGAA

GCGCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCG

CAGATTCTGCAGCAGGCCGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTCCCGCAGAAC

GTGCTGAGCCTGTTAGCGATGGAATGGGAAAATATCCCTAGCATCCAATCTCGCGGTCTG

TTCGGTGCTATTGCTGGCTTCATCGAGGAATGGGAGAACATCCCATCTATTCAGTCTCGC

GGCCTGTTTGGTGCGATCGCGGGTTTTATTGAGTGATAAGCTTAGCCCAAGG

SEQ ID NO：355 STF2.1xH1C1

ATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAA

TCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGC

GCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGT

CTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGC

GCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCT

AACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTG

AACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAG

GACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTG

AAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCAGAAAGCGTATGAT

GTGAAAGATACAGCAGTAACAACGAAAGCTTATGCCAATAATGGTACTACACTGGATGTA

TCGGGTCTTGATGATGCAGCTATTAAAGCGGCTACGGGTGGTACGAATGGTACGGCTTCT

GTAACCGGTGGTGCGGTTAAATTTGACGCAGATAATAACAAGTACTTTGTTACTATTGGT

GGCTTTACTGGTGCTGATGCCGCCAAAAATGGCGATTATGAAGTTAACGTTGCTACTGAC

GGTACAGTAACCCTTGCGGCTGGCGCAACTAAAACCACAATGCCTGCTGGTGCGACAACT

AAAACAGAAGTACAGGAGTTAAAAGATACACCGGCAGTTGTTTCAGCAGATGCTAAAAAT

GCCTTAATTGCTGGCGGCGTTGACGCTACCGATGCTAATGGCGCTGAGTTGGTCAAAATG

TCTTATACCGATAAAAATGGTAAGACAATTGAAGGCGGTTATGCGCTTAAAGCTGGCGAT

AAGTATTACGCCGCAGATTACGATGAAGCGACAGGAGCAATTAAAGCTAAAACTACAAGT

TATACTGCTGCTGACGGCACTACCAAAACAGCGGCTAACCAACTGGGTGGCGTAGACGGT

AAAACCGAAGTCGTTACTATCGACGGTAAAACCTACAATGCCAGCAAAGCCGCTGGTCAT

GATTTCAAAGCACAACCAGAGCTGGCGGAAGCAGCCGCTAAAACCACCGAAAACCCGCTG

CAGAAAATTGATGCCGCGCTGGCGCAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTA

CAAAACCGTTTCAACTCTGCTATCACCAACCTGGGCAATACCGTAAACAATCTGTCTGAA

GCGCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCG

CAGATTCTGCAGCAGGCCGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTCCCGCAGAAC

GTGCTGAGCCTGTTAGCGATGGAATGGGAAAATATCCCTAGCATCCAATCTCGCGGTCTG

TTCGGTGCTATTGCTGGCTTCATCGAGTGATAAGCTTAGCCCAAGG

SEQ ID NO：356 STF2.4xH1C1

ATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAACAAA

TCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGC

GCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAACATCAAAGGT

CTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGC

GCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCT

AACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTG

AACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAG

GACAACACCCTGACCATCCAGGTTGGCGCCAACGACGGTGAAACTATCGATATCGATCTG

AAGCAGATCAACTCTCAGACCCTGGGTCTGGACTCACTGAACGTGCAGAAAGCGTATGAT

GTGAAAGATACAGCAGTAACAACGAAAGCTTATGCCAATAATGGTACTACACTGGATGTA

TCGGGTCTTGATGATGCAGCTATTAAAGCGGCTACGGGTGGTACGAATGGTACGGCTTCT

GTAACCGGTGGTGCGGTTAAATTTGACGCAGATAATAACAAGTACTTTGTTACTATTGGT

GGCTTTACTGGTGCTGATGCCGCCAAAAATGGCGATTATGAAGTTAACGTTGCTACTGAC

GGTACAGTAACCCTTGCGGCTGGCGCAACTAAAACCACAATGCCTGCTGGTGCGACAACT

AAAACAGAAGTACAGGAGTTAAAAGATACACCGGCAGTTGTTTCAGCAGATGCTAAAAAT

GCCTTAATTGCTGGCGGCGTTGACGCTACCGATGCTAATGGCGCTGAGTTGGTCAAAATG

TCTTATACCGATAAAAATGGTAAGACAATTGAAGGCGGTTATGCGCTTAAAGCTGGCGAT

AAGTATTACGCCGCAGATTACGATGAAGCGACAGGAGCAATTAAAGCTAAAACTACAAGT

TATACTGCTGCTGACGGCACTACCAAAACAGCGGCTAACCAACTGGGTGGCGTAGACGGT

AAAACCGAAGTCGTTACTATCGACGGTAAAACCTACAATGCCAGCAAAGCCGCTGGTCAT

GATTTCAAAGCACAACCAGAGCTGGCGGAAGCAGCCGCTAAAACCACCGAAAACCCGCTG

CAGAAAATTGATGCCGCGCTGGCGCAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTA

CAAAACCGTTTCAACTCTGCTATCACCAACCTGGGCAATACCGTAAACAATCTGTCTGAA

GCGCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCG

CAGATTCTGCAGCAGGCCGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTCCCGCAGAAC

GTGCTGTCTCTGTTAGCGGGTTGTGGTTCCGAGTGGGAAAATATTCCGTCTATCCAGAGC

CGTGGTCTGTTCGGCGCAATTGCTGGCTTCATTGAAGAATGGGAAAACATCCCGTCCATC

CAGAGCCGTGGCCTGTTCGGCGCCATTGCTGGTTTCATCGAGGAATGGGAAAACATTCCG

TCCATCCAGTCCCGCGGTCTGTTTGGCGCTATCGCCGGTTTCATTGAGGAATGGGAAAAT

ATCCCTTCCATCCAGTCTCGTGGTCTGTTCGGCGCGATTGCAGGCTTTATCGAATCTGGT

TGCTAATAAGCTAAGC

The colibacillary bacterial lipoprotein of SEQ ID NO:357

MKATKLVLGAVILGSTLLAGCSSNAKIDQLSSDVQTLNAKVDQLSNDVNAMRSDVQAAKDDAARANQ

RLDNMATKYRK

SEQ ID NO:358 H1C1 native peptides

SLWSEENIPSIQSRGLFGAIAGFIEE

Embodiment 15: flagellin-M2e fused protein

M2e has conservative in most influenza A hypotypes (also being called " Strain " in this paper).M2e is the proteic 24-amino terminal of M2 proteic at least a portion, especially M2 (also being called " functional domain outside the born of the same parents " in this paper).The outer functional domain of M2 born of the same parents is compared to HA (about 566 aminoacid) and NA (about 469 aminoacid), for going up less aminoacid sequence (24 aminoacid) relatively.The M2e sequence of exemplary birds influenza A separated strain is different with human separated strain, but between the birds separated strain, have high conservative (referring to, for example SEQ ID NOS:544-556,570 and 573-578).Produce four tandem copies of M2e, with the carboxyl terminal fusion of flagellin STF2 (total length or STF2 hinge region are lacked).The STF2 that does not contain hinge region also is called " STF2 Δ " in this paper.

The structure of fused protein

Synthesis type 4xM2e sequence (4 sections consecutive 24 aminoacid sequences) merges with the carboxyl terminal of STF2, is according to following the structure.The pET24A carrier is available from Nova gold, Santiago, CA.This strategy is to use (Stratagene, La Jolla, CA available from Shi Tajin Www.stratagene.com) no seam clone test kit (catalog number 214400), (cover Luo Gongyuan, CA) finish by DNA 2.0Inc..The gene of encoding fusion protein is to exist among the pDrive 4xM2e G00448, and is used for holding the insert preparation of amalgamation and expression construct as pcr template for the C-that makes up with STF2.Synthesis type 4xM2e construct pDrive4xM2e G00448 is used for as (La Qiaola, no seam CA) is cloned the listed PCR of test kit (catalog number 214400) as template available from Shi Tajin.From then on the desired product that increases comprises that 318bp and restriction enzyme sites incorporate the oligonucleotide of this insert that is used to increase into.This program is as follows:

The PCR condition

The pDrive 4xM2e G00448 of 1 μ L-20ng

The 10x clone Pfu polymerase buffer of 5 μ L

The 40mM dNTP mix of 1 μ L

The forward primer 4xM2eforbsl of 1 μ L-10pmol

The reverse primer 4xM2erevwsto of 1 μ L-10pmol

40μL ddH ₂O

Before the beginning thermal cycle, the PfuTurbo archaeal dna polymerase of beginning with 1 μ L adds.

The 4xM2eforbs1 primer sequence:

5’-CGCTCTTCAMTGAGCTTGCTGACTGAGGTTGAGACCCCGATTC(SEQ ID NO：566)

The 4xM2erevwsto primer sequence:

5’-CGCTCTTCACGCTTATTATCTAGACGGGTCTGAGCTATCGTTAGAGCGAG(SEQ ID NO：567)

This reaction is that following (Vaasa nurse (Waltham) MA) circulates in Thermo Hybaid PxE thermo cycler.

Initial cycle

Temperature	Time
			95℃	3 minutes
65℃	1 minute
		72℃	1 minute

Follow-up nine circulations

Temperature	Time
			95℃	45 seconds
65℃	35 seconds
		72℃	1 minute

Add in each reaction following this moment.

The 10x clone Pfu polymerase buffer of 5 μ L

The 5-methyl dNTP mix of 1 μ L

44μL ddH ₂O

Then repeat following thermal cycle five times.

Temperature	Time
			95℃	45 seconds
65℃	35 seconds
		72℃	1 minute

100 μ L products are passed through to add the TE buffer, and volume is brought to 300 μ L.The product that is become is extracted once through phenol chloroform (Yin Weituo King Company, Ka Sibeide, CA-catalog number 15593-031), and chloroform extraction once.Then amplified production is passed through to add 30 μ L sodium-acetate buffer pH 5.2, and 750 μ L of, 100% ethanol carries out alcohol precipitation.The DNA precipitation is cleaned and air-dry ten minutes with 300 μ L70% ethanol, and then be suspended in the 50 μ L TE buffer.

Carrier S TF2 is in the amplification of pET24

With before made up the pET24a/STF2.M2e construct be used for, as (La Jolla, the listed PCR of no seam clone's test kit (catalog number 214400) CA) is as template available from Shi Tajin.From then on the desired product that increases comprises that whole pET24 plasmid adds the STF2 sequence, but does not comprise the single M2E copy that exists in this construct.This program is as follows:

The PCR condition

The STF2.M2E pET22-2 of 1 μ L-20ng

The 10x clone Pfu polymerase buffer of 5 μ L

The 40mM dNTP mix of 1 μ L

The primer 4xMECpET24 of 1 μ L-10pmol

The primer 4xM2eC-STF2 of 1 μ L-10pmol

40μL ddH ₂O

Before the beginning thermal cycle, begin following adding:

The PfuTurbo archaeal dna polymerase of 1 μ L

The 4xMECpET24 primer sequence:

5’-GCTCTTCAGCGGCTGAGCAATAACTAGCATAACCCCTTGGG(SEQID NO：568)

The 4xM2eC-STF2 primer sequence:

5’-CGCTCTTCACAGACGTAACAGAGACAGCACGTTCTGCGG(SEQ IDNO：569)

This reaction is that following (the Vaasa nurse MA) circulates in Thermo Hybaid PxE thermo cycler.

Initial cycle

Temperature	Time
			95℃	3 minutes
65℃	1 minute
		72℃	18 minutes

Follow-up nine circulations

Temperature	Time
			95℃	45 seconds
65℃	35 seconds
		72℃	18 minutes

Add in each reaction following this moment.

The 10x clone Pfu polymerase buffer of 5 μ L

The 5-methyl dNTP mix of 1 μ L

44μL ddH ₂O

Then repeat following thermal cycle five times.

Temperature	Time
			95℃	45 seconds
65℃	35 seconds
		72℃	18 minutes

100 μ L products are passed through to add the TE buffer, and volume is brought to 300 μ L.The product that is become is extracted once through phenol chloroform (Yin Weituo King Company, Ka Sibeide, CA-catalog number 15593-031), and chloroform extraction once.Then amplified production is passed through to add 30 μ L sodium-acetate buffer pH 5.2, and 750 μ L of, 100% ethanol carries out alcohol precipitation.The DNA precipitation is cleaned and air-dry ten minutes with 300 μ L, 70% ethanol, and then be suspended in the 50 μ L TE buffer.

The decomposition of carrier and insert amplification and engaging

It is as follows to equip the Eam 1104I analyte that is used for carrier and insert respectively:

The amplified production of 30 μ L behind alcohol precipitation

The general buffer of the 10x of 5 μ L (investing no seam clone test kit)

4 μ L Eam 1104I restriction endonucleases (investing no seam clone test kit)

11μL ddH ₂O

Mix and cultivated analyte is gentle,, carry out following joint reaction (reagent invests no seam and clones test kit) carrier and the insert product of preparation as described above in 37 ℃ of next hours:

Add the composition that is listed in regular turn:

9μL ddH ₂O

The 4xM2e that decomposes through Eam 1104I of the 5 μ L insert that increased

The STF2.M2E pET22-2 that decomposes through Eam 1104I of 5 μ L is amplification vector

2 μ L 10x engage buffer

2μL 10mM rATP

1 μ L T4 DNA joining enzyme (1:16 dilutes from reserve)

1 μ L Eam 1104I restriction endonuclease

To engage reaction and leniently mix, and cultivate 30 minutes down in 37 ℃.Then jointer is stored on ice, up in being converted into the competent cell (Shi Tajin catalog number 200314) of XL-10 on the same day after a while.

Jointer is converted in the competent cell of XL-10

With microcentrifugal tube (Eppendorf tubes) cooling ten minutes, the competent cell of XL-10 (Shi Tajin catalog number 200314) is placed on ice thaw simultaneously.

To be competent at cell and be used for each jointer from every pipes of branch 50 μ L such as deposit test tubes.

2 μ L follow the appended beta-mercaptoethanol reserve of XL-10 cell.

This mixture in cultivating ten minutes, is followed per 2 minutes gentle mixing on ice.To not have the seam clone and engage reaction (4 μ l) adding, gentle vortex is cultivated then in 30 minutes on ice.With centrifuge tube under 42 ℃ in water-bath heat shock 35 seconds.With centrifuge tube in cultivating at least two minutes on ice.(400 μ L) adds to cell with the SOC culture medium, and follows to stir under 37 ℃ and cultivated one hour.

With two tablets of LB agar kanamycin (50 μ g/mL) flat board, be used to be coated with the transformant of 200 μ L and 10 μ L, and make its grow overnight.

Kalamycin resistance clone's screening

The reorganization candidate strain that will be used for preparation in a small amount grows in the Luria fluid medium that contains kanamycin (25 μ g/mL), and uses QIAprep Spin Miniprep Kit (all West Asias of Kui Erjin (Qiagen), CA catalog number 27106) to extract.Restriction endonuclease (New England's biology laboratory (NewEngland Biolab) is passed through in the pure strain of candidate, Bei Fuli, MA) screening, and positive clone strain grown in the Luria fluid medium that contains kanamycin (25 μ g/mL), and use Kui Er gold HiSpeed Plasmid Midi Kit (catalog number 12643) to extract.GENEWIZ is delivered in these pure strains, and (northern cloth Southwick NJ) carries out sequencing.

The manufacturing of STF2.4xM2e fusion rotein and purification

STF2.4xM2e existed among escherichia coli BLR (DE3) the pLysS host (Nova gold, Santiago, CA, catalogue #69053) from the glycerol reserve recover, and be amplified to 5L.Cell is grown in the LB culture medium that contains 15 μ g/ml kanamycin and 12.5 μ g/ml tetracyclines to OD ₆₀₀=0.4, and cultivate down in 37 ℃ with 1mMIPTG and to induce in 3 hours.Cell is collected by centrifugal (7000rpm x 7 minutes in Sorvall RC5C centrifuge), and resuspending is in 2x PBS, 1% glycerol, 1 μ g/ml DNAseI, 1mM PMSF, protease inhibitor cocktail and 1mg/ml lysozyme.Suspension is made cytolysis by little liquefier.With lysate centrifugal (in Beckman Optima L supercentrifuge, in 45,000g next hour), so that solubility is separated with inclusion body.Protein is detected in solvable and soluble part by SDS-PAGE.

Existing down in high salt soluble part, application of sample reduces DNA, endotoxin and other impurity to agarose Q resin via batch part method.To contain to some extent the proteinic merchantable thing application of sample of interest to 30ml Q agarose tubing string (Ai Moxun bioscience).Use linear gradient elution to go out conjugated protein from buffer A to buffer B.(buffer A: 100mM Tris-Cl, pH 8.0.Buffer B: 100mM Tris-Cl, 1M NaCl, pH 8.0).The protein that eluting is gone out further uses 45ml sucrose Q tubing string purification, and it can provide in order to resolve the proteinic big resolution of mixing.With conjugated protein so that (buffer A: 100mM Tris-Cl, pH 8.0 from buffer A to buffer B.Buffer B: 100mM Tris-Cl, 1M NaCl, pH 8.0) linear gradient elution go out.

Use Superdex-200 gel permeation chromatography art, finish proteinic final purification.This tubing string is with 100mM Tris, and 150mM NaCl and 1% glycerol add the 1%Na-deoxycholic acid to launch, to remove LPS.Use is to containing 50mM Tris, and the buffer of 100mM NaCl and 1% glycerol is finished dialyzed overnight, carries out buffer-exchanged to remove the Na-deoxycholic acid.Use MicroBCA protein analysis reagent test kit (Pierre's Si biotechnology) to measure protein concentration.The purified preparation of STF2.4xM2e presents single band with Kao Maxi dyeing, and it shows that on 12% sds page having molecular weight is about 64kDa.

Embodiment 16: the performance and the purification of flagellin (STF2 and STF2 Δ) the fused protein construct of the outer functional domain sequence of coding influenza A M2 born of the same parents

Derive from several human influenza A virus strains, and poultry derived total M2e sequence is through being shown in SEQID NOS:544-556,570 and 573-578.For helping clone the M2e sequence, produce two kinds of support card caskets that respectively contain multiple cloning site (MCS) then, pMT/STF2 and pMT/STF2 Δ (referring to Figure 17 A and 17B).For producing pMT/STF2, the 1.5kb gene of the total length of will encoding salmonella typhimurium flagellin fljb the 2nd type (or STF2), merge to the pMTBIP/V5-His carrier (Yin Weituo King Company, Ka Sibeide, Ig CA) conjugated protein (BIP) secretion signal that supply to be expressed in fruit bat.The BiP sequence is to be included in 5 of this construct ' end, as the secretion signal that supplies to be expressed in fruit bat.Get the 4xM2e gene with what represent H1, H2 and H3 consensus sequence through chemosynthesis, be cloned among the MCS of pMT/STF2 and produce pMT/STF2.4xM2e (H1).

Prophesy property ground uses similar strategy, clone two kinds of H5-connection M2e sequences, SLLTEVETPTRNEWECRCSDSSDP (SEQ ID NO:553) (A/ Vietnam/1203/2004) and SLLTEVETLTRNGWGCRCSDSSDP (SEQ ID NO:552) (A/ Hong Kong/156/97).Four series connection that will contain prophesy property ground specified H5-connection M2e sequence repeat copy, the gene clone that gets through codon-optimization, with chemosynthesis go among the pMT/STF2 and produce STF2.4xM2e respectively (H5VN) and STF2.4xM2e (H5HK).For generation contains the construct of multiple M2e form, satisfy with allos 4xM2e sequence prophesy property ground insert described elementary construct wherein one.

" heterologous sequence " is used for this paper and means the sequence that derives from different plant species.For example, the H1 sequence is the human sequence, and the H5 sequence is the birds sequence.Therefore, H1 and H5 sequence be heterologous sequence (for example, SLLTEVETPTRNEWESRSSDSSDPLESLLTEVETPTRNEWESRSSDSSDPESSLLT EVETPTRNEWESRSSDSSDPGSSLLTEVETPTRNEWESRSSDSSDP (SEQID NO:597)), coded by tctctgctgactgaagtagaaactccaacgcgtaatgaatgggaatcccgttctag cgactcctctgatcctctcgagtccctgctgacggaggttgaaaccccgacccgca acgagtgggaaagccgttcctccgattcctctgatccggagagcagcctgctgacc gaggtagaaaccccgacccgtaatgagtgggaatctcgctcctctgattcttctga cccgggatcctctctgctgaccgaagtggagactccgactcgcaacgaatgggaga gccgttcttctgactcctctgacccg (SEQ IDNO:598).

Elementary construct comprises the molecule pattern (for example, STF2, STF2 Δ) of at least a pathogen-connection, and at least a portion (for example, M2e such as SEQ ID NOS:510 and 544) of at least a conformability memebrane protein.If there are more than one conformability memebrane proteins to exist in the elementary construct, then this conformability memebrane protein is to be derived from same species.

The allos construct comprises at least two kinds of conformability memebrane proteins, and for example H1 (mankind) and H5 (birds) are as in SEQ ID NOS:583 and 584.

For producing the pMT/STF2 Δ, then will cross over the hypervariable region disappearance of amino acid/11 70 to 415 of the total length flagellin gene of SEQ ID NO:499, and displacement helps TLR5 to summon the effect of necessary amino and carboxyl with one section through design, short (ten amino acid) flexible peptide connexon (GAPVDPASPW, SEQID NO:594).From then on the expressed protein of construct still keeps effective TLR5 activity, no matter and its be separately or with the test antigen amalgamation and expression.Therefore, prophesy property ground serves as that the basis produces second series M2e construct with the pMT/STF2 Δ.To under room temperature, grow in replenish with 10% FBS with antibiotic perhaps how the fruit bat Dmel-2 cell in the De Shi culture medium (because of the Vito gold; Ka Sibeide CA), uses Cellfectin reagent (because of the Vito gold), foretells that according to the explanation of manufacturer property ground changes infection with aforesaid construct.In change infecting the back twenty four hours, prophesy property ground with cell with 0.5mM CuSO ₄, in the culture medium that lacks FBS, also cultivate in addition again and induced in 48 hours.Prophesy property ground is from collecting conditioned medium (CM) through inductive culture, and by SDS-PAGE and use the western blot analysis screening protein expression of anti--flagellin and anti--M2e specific antibody.By homogeneity, TLR biological activity, the antigenicity of elisa assay fusion rotein, and prophesy property ground carries out in vivo mice study and analyzes its immunogenicity.

Embodiment 17: the structure and the performance of flagellin-hemagglutinin (HA)

From deriving from the genomic DNA of interior tissue laboratory strain PR8 (for the attenuation derivant of a kind of A/ Puerto Rico/8/34), separate the gene (SEQ ID NO:565, coding SEQ ID NO:564) of coding HA.This gene fusion to the STF2 Δ card casket in being implemented in the pPICZ Δ in advance, is produced STF2 Δ .HAPR8 (SEQ ID NO:560, coding SEQ ID NO:559) (referring to Figure 42).Purified recombinant protein in BALB/c mouse, is tested its immunogenicity and effect.The gene of the H5N1 of composite coding A/ customized Vietnam/1203/04 Strain, and merge to STF2 Δ card casket and produce STF2 Δ .HAH5 (SEQ ID NO:558, coding SEQ ID NO:557).With the mankind and birds HA construct be converted into pichia pastoris phaff bacterial strain GS115 and X-33 (Yin Weituo King Company, Ka Sibeide, CA) in.With selected pure strain by fraction on the SDS-PAGE gel with the blue dyeing of Kao Maxi, and use the western blot analysis screening protein expression of anti--HA and anti--flagellin antibody.

The generation of embodiment 18:PAM3CYS fusion rotein

With the amino terminal serine residue of M2e (SEQ ID NO:544), be coupled to three palmityl cysteine (Pam3Cys) parts with chemical mode through this peptide.The structure of fusion rotein (Pam3Cys.M2e) is shown in Figure 39.The chemistry of Pam3Cys.M2e is called [palmityl-Cys ((RS)-2,3-two (palmityl oxygen base)-propyl group)-Ser-Leu-Leu-Thr-Glu-Val-Glu-Thr-Pro-Ile-Arg-Asn-Glu-Trp-Gly-Ser-Arg-Ser-Asn-Asp-Ser-Ser-Asp-Pro-OH acetate].The molecular weight of Pam3Cys.M2e is 3582.3 dalton.

Pam3Cys.M2e is to use, with Fmoc-strategy (Hu Ben-Weir (Houben-Weyl), 2004 of suitable foundation.Synthetic (the Synthesis of peptides andpeptidomimetics) of peptide class and peptide mimics, volume 22, George's Tim is published (Georg Thieme Verlag Stuttgart), NY) be that the solid-phase peptide synthetic method on basis is synthesized.The synthesis flow of Pam3Cys.M2e and manufacture method are with following flowcharting.Pam3Cys.M2e is fused protein (getting through chamical binding), and also is called " lipid peptide " in this paper.

Synthetic first step comprises that solid-phase peptide is synthetic.Be combined on the chlorination H-Pro-2-chlorine trifluoromethyl resin by the synthetic aminoacid sequence of solid-phase peptide Pam3Cys.M2e.This resin is well suited for being used for forming the peptide class with the Fmoc-strategy.Peptide chain is to increase by continuous coupling amino acid derivant.Before each coupling step is that a Fmoc-goes to protect step, and separating this two step all is to finish by the repeated washing resin.Behind last amino acid derivativges of coupling, carry out last Fmoc-and go to protect step.At last, peptide resin is cleaned and drying under decompression.In solid-phase peptide between synthesis stage, carry out the color indicator test of each step, whether complete with monitoring Fmoc-cracking and follow-up amino acid derivativges coupling.

The synthetic stage 2 comprises the coupling of Pam3Cys-OH.Pam3Cys-OH is existed down in I-hydroxybenzotriazole (HOBt), and with N, N '-dichloro hexyl-carbonyl imidodicarbonic diamide (DCCI) activates in advance.The solution that is become is filtered and adds to peptide resin.When the response time finishes, peptide resin is cleaned and drying under decompression.Carry out the color indicator test, with the coupling of management and control Pam3Cys-OH.

The synthetic stage 3 comprises from pitch shake and solving, and it comprises the cracking of Side chain protective group.Peptide resin is handled with trifluoroacetic acid (TFA).Product is settled out and lyophilization from reactant mixture.

The synthetic stage 4 comprises, HPLC carries out purification by preparation type anti-phase.To derive from the crude material in stage 3, on the anti-phase tubing string that uses the TFA system, carry out purification with preparation HPLC.Collect stream part, compile by analytical type HPLC inspection and with it.Will from TFA carry out compile the lyophilization of stream part.

The synthetic stage 5 comprises, precipitates under EDTA exists.With deriving from the purifying substance in stage 4, be settled out from the EDTA aqueous solution.Drying filters out product and reduce pressure down.

The synthetic stage 6 comprises the ion-exchange chromatography art.Making the final stage of Pam3Cys.M2e, is that it is exchanged into acetate by ion exchange from trifluoroacetate.The material application of sample that derives from the stage 5 to the ion exchange tubing string, and is carried out eluting with acetic acid.By each stream of thin layer chromatography art inspection part, and with make up contain product stream part filter and lyophilization and produce end product.

Pam3-Cys-OH=palmityl-Cys ((RS)-2,3-two (Petiolus Trachycarpi acyloxy)-propyl group)-OH

By RP-HPLC, the pureness specifications of Pam3Cys.M2e drug substance is 〉=80%.This specification is based on three batches and organizes from the GMP with a collection of M2e-intermedium resin, and non--GMP of obtained Pam3Cys.M2e organizes the purity that reaches.The purity of non--GMP group of these three crowdes of Pam3Cys.M2e is 80.2%, 80.3% and 80.8%, corresponds respectively to D.001.Pam3Cys.M2e, D.002.Pam3Cys.M2e reaches D.003.Pam3Cys.M2e.

Embodiment 19: immunogenicity

Materials and methods

Synthetic and the purification of PAM3CYS.M2E

Pam3Cys.M2e is synthetic and Bachem by Genemed, and using as the aforementioned, solid phase synthesis process and FMOC chemical preparation get.Use mass spectral analysis to determine the molecular weight of end product.

Endotoxin analysis

Use the QCL-1000 LAL test kit (BioWhittaker#50-648U) that quantitatively develops the color, be used for the explanation of micro plate method, measure the endotoxin concns of STF2.4xM2e and Pam3Cys.M2e according to manufacturer.

The TLR5 biological activity is analyzed

The HEK293 cell is expressed TLR5 inherently, and in the reaction to the TLR5 citation, secretes several soluble factors (comprising IL-8).With HEK293 cell inoculation (50,000 cells/well) in the micro plate of 96-hole, and test protein added and cultivate and spend the night.Next day, collect conditioned medium, be transferred to clean 96-hole micro plate, and freezing down in-20 ℃.After waiting to thaw, in the antibody that uses Anti-Human's class-IL-8 to cooperate to (Pierre Si; #M801E and #M802B) sandwich ELISA, according to the existence of IL-8 in the explanation analysis condition culture medium of manufacturer.(FARCyte Ai Moxun) measures optical density (OD) to use the micro plate spectrophotometer.The result is through being reported to, with the standard curve conclusion of IL8 in this analysis measured the pg number of IL8 among every ml.

The TLR2 biological activity is analyzed

RAW264.7 cell (ATCC) is expressed TLR2, and in the reaction to the TLR2 citation, secretes several soluble factors (comprising TNF α).With RAW264.7 cell inoculation (50,000 cells/well) in the micro plate of 96-hole, and test protein added and cultivate and spend the night.Next day, collect conditioned medium, be transferred to clean 96-hole micro plate, and freezing down in-20 ℃.After waiting to thaw, in the sandwich ELISA of the antibody that uses anti--mice TNF α to cooperate, according to the existence of TNF α in the explanation analysis condition culture medium of manufacturer to (Pierre Si).(FARCyte Ai Moxun) measures optical density (OD) to use the micro plate spectrophotometer.The result is through being reported to, with the standard curve conclusion of TNF in this analysis measured the pg number of TNF among every ml.

Mouse immune originality

Use the about 6-8 female BALB/c mouse in age in week (national cancer association).With 5 to 10 every group of mice group, and, exempt from subcutaneous causing in the both sides of afterbody base portion in the 0th and 21 day STF2.4xM2e or Pam3Cys.M2e fusion rotein with specified concentration.With mice puncture blood collecting after the 10th day (elementary) and 28 days (the short liter) passes through eye socket.And condense and the centrifugal serum of gathering by will there not being the heparinemia fluid samples.

The mice serum TPPA

Measure M2e-specific IgG concentration by ELISA.(Costar (Cat#9018) is healthy and free from worry, and NY) under 4 ℃, the 5 μ g/ml solution that are dissolved in PBS with 100 μ l/ hole M2e peptides apply and spend the night with 96-hole elisa plate.With the analysis dilution buffer liquid (ADB of plate with 200 μ l/ holes; BD Fa Minggen) under room temperature, blockaded one hour.Plate is cleaned three times in the PBS (PBS-T) that contains 0.05% (v/v) Tween 20.Serum is added (100 μ l/ hole) in the diluent of ADB, and plate cultivation under 4 ℃ is spent the night.Plate is cleaned three times with PBS-T.To be diluted in the mountain horseradish peroxidase of ADB, or the HRP-labelled goat anti--mouse IgG antibody (Jackson's immunochemistry) adds (100 μ l/ hole), and plate cultivated under room temperature 1 hour.Plate is cleaned three times with PBS-T.TMB Ultra substrate to be added (3,3 ', 5,5 '-tetramethyl benzidine; And behind the monitoring color colour generation, (Dole breathes out, and NC) measures O.D.450 on the micro plate spectrophotometer in Tecan Farcyte Pierre Si).

The rabbit immunogenicity

Use about 13-17 male and female NZW rabbit (Ke Fansi studies product) in all ages.With rabbit divide into groups 3 every group male with 3 female, and in the 0th and 21 and 42 day,, on mutual thigh, cause and exempt from i.m. with the STF2.4xM2e peptide or the Pam3Cys.M2e fusion rotein of specified concentration.With animal in the-1 day (preceding blood sampling), 14 (elementary) and (the short liter) blood sampling in 28 and 42 days.And by sample being condensed and the centrifugal serum of gathering.

The rabbit anteserum TPPA

Measure M2e-specific IgG concentration by ELISA.96-hole elisa plate under about 4 ℃, is dissolved in PBS (5 μ g/ml) with 100 μ l/ hole M2e peptides and applies and spend the night.With the analysis dilution buffer liquid (ADB of plate with 200 μ l/ holes; BD Fa Minggen) under room temperature, blockaded one hour.Plate is cleaned in PBS-T three times.Serum is added (100 μ l/ hole) in the diluent of ADB, and plate cultivation under 4 ℃ is spent the night.Plate is cleaned 3x with PBS-T.Use the conjugated goat of HRP-anti--rabbit igg (Jackson's immunochemistry) detects bonded IgG.Plate is cleaned three times with PBS-T.TMB Ultra substrate to be added (Pierre Si), and behind the monitoring color colour generation, on molecular device Spectramax micro-spectrophotometer, measure O.D.450.The result is reported to, and the O.D.450 value of reading that causes after exempting from by each animal deducts the O.D.450 value of reading of taking a blood sample before each animal, and measure Delta O.D..

The BALB/C mice efficacy models

In representational experiment, be to buy about 5-6 female BALB/c mouse in all ages (every group of 10-20 only), and make it adapt to a week.Give through allocating by s.c. injection throwing in the fused protein of PBS or other suitable concoction.Mice caused in the 0th and 14 day exempt from.In the 21st day by eye socket after the puncture serum of gathering, and by ELISA assessment M2e specific IgG.Mice is thrown the quite characterization mice adaptation influenza A virus strain give 1xLD90 by intranasal, A/ Puerto Rico/8/34 (H1N1) and excite.Survival and the body weight loss of investigating animal every day reach 14 days.Its body weight is lost 30% mice with the euthanasia sacrifice compared to the initial body density current, and will sacrifice day entry for and dead day.Efficacy data is to report with the time-to-live.

The result

Biological activity in vitro

These analyses are expressing the cell of suitable TLR, and screening its in the reaction that TLR is caused, make the ability of IL8 or TNF-α.In Figure 52, be after stimulating the positive HEK293 cell of TLR5, the ability that assessment STF2.4xM2e (■) or STF2.OVA (zero) can stimulate TLR5 dependency IL8 to make.The result represents that this two fusion rotein all stimulates IL8 to make in dosage dependence mode, and the activity of PAMP still remaines in the fusion rotein.

Similarly, after stimulating the positive RAW264.7 cell of TLR2, assess at Pam3Cys.M2e.In Figure 51, experimental group is: the inhibitor that known endotoxin, LPS (as positive controls (◆)), LPS add endotoxin polymixin B (PMB) adds PMB (√) as negative control group (zero), free state Pam3Cys as positive controls (■), the free state Pam3Cys of TLR2 citation, and Pam3Cys.M2e (◆) and Pam3Cys.M2e add PMB ().The result shows that Pam3Cys.M2e has similar active collection of illustrative plates to free state TLR2 part Pam3Cys.Add polymixin B (PMB) and can not reduce its activity, expression does not have or has only low contaminated with endotoxins.

PAMP and antigenic physics binding increase immunogenicity

Use immunogenic mouse model, the chemical coupling of Pam3Cys and M2e can increase the M2e immunogenicity of antigens, when compared to sending the M2e peptide separately or sending jointly with free state Pam3Cys.In the experiment shown in Figure 52, with each group mice in the 0th and 21 day, with PBS as negative control group (.*), free state TLR2 part, Pam3CSK-4 ((), M2e peptide (zero), free state Pam3CSK-4 mix with M2e peptide (√) or are called Pam3.M2e (◆) with the fusant of Pam3Cys and M2e and cause and exempt from separately.With sending M2e peptide suitably the ear ratio keep inconstant.In the 28th day serum of gathering, and by elisa assay M2e-specific antibody titres.The result shows the chemical coupling (Pam3Cys.M2e) of Pam3Cys and M2e, produces detectable at the antigenic serum antibody response of M2e.

Physics binding between TLR5 part STF2 and antigen is to use pattern antigen ovalbumin (OVA) to prove.Mice accept with STF2, OVA, STF2.OVA fusion rotein, STF2+OVA mixture or separately the single s.c. of PBS cause and exempt from.Calculate dosage with every group of STF2 and OVA that sends 12 μ g of a great deal of.Behind the Yu Qitian, the serum and of gathering by ELISA check OVA-specific antibody.Data description in Figure 53, the IgG1 under the 1:100 serum dilution tires.These results prove that the physics binding between TLR5 part and antigen causes the suitableeest in vivo immunogenicity.

Have more immunogenicity through the more known adjuvant of the antigen of PAMP binding

With every group of 5 BALB/c mouse in the 0th and 14 day, with the Pam3Cys.M2e (◆) of 30 μ g, the M2e of 22.5 μ g (it equates with the not ear number of M2e among the 30 μ g Pam3Cys.M2e) (), 22.5mg is adsorbed in the M2e (√) of known adjuvant Alumen or 25mg recombinant protein STF2.4xM2e (■) and causes and exempt from.Comprise that a winding is subjected to PBS as negative control group (zero).Behind second time dosage, collected serum in 7 days, and by ELISA assessment M2e specific IgG.The result who is shown in Figure 54 points out, the independent immunogenicity of M2e is poor, because of it can't cause the antibody titer that is higher than background value.Known adjuvant Alumen provides the immunoreation of appropriateness increase to M2e.Yet, provide immunogenic maximum the enhancing through the M2e of PAMP binding construct.These results point out the part binding of the conformability memebrane protein of PAMPs and influenza virus protein, can cause by the more effective immunoreation of known adjuvant Alumen institute's causer.

Dosage and immunogenicity

Carry out the effect of dosage range research with further analysis Pam3Cys.M2e and STF2.4xM2e.For STF2.4xM2e, be with BALB/c mouse in the 0th and 14 day, cause the STF2.4xM2e diluent of exempting from scope from 0.25 to 25 μ gSTF2.4xM2e at every turn and cause and exempt from.Prefix D002 is meant particular lot part of the STF2.4xM2e that is used for this experiment, and R-028 means the hospital of the STF2.4xM2e that is used for this experiment with reference to batch part.Cause in for the last time and to exempt from (the 21st day) back seven days the mice blood sampling, and by the reaction of ELISA assessment M2e-specific IgG.The result who is shown in Figure 55 proves, the dosage that at every turn causes the STF2.4xM2e that exempts from low as 0.25 μ g causes exempts from, but brings out the M2e-specific IgG of detection limit, falls within about 2.5 to the scope of about 25 μ g in the dose,optimum of mice.

For Pam3Cys.M2e, be with BALB/c mouse in the 0th and 14 day, cause and exempt to cause the Pam3Cys.M2e that exempts from 0.05 to 30 μ g at every turn.Cause in for the last time and to exempt from (the 21st day) back seven days the mice blood sampling, and by the reaction of ELISA assessment M2e-specific IgG.The result who is shown in Figure 56 proves, causes with the concentration of low as 0.05 μ gPam3Cys.M2e and exempts from, but bring out the M2e-specific IgG of detection limit, and be about 30 μ g in this research for dose,optimum of mice.

Immunogenicity in most mouse species

In the immunogenicity that comprises BALB/c (●), C57BL/6 (■), CB6/F1 (◆), DBA/2 (▲), Cr:NIH (Switzerland) assessment Pam3Cys.M2e (X) and in most mouse species of C3H/HeN (*).Every group of 5 BALB/c mouse of each strain in the 0th and 14 day, are caused and exempt to cause the Pam3Cys.M2e that exempts from 30 μ g at every turn.In the 21st day serum of gathering, and by ELISA assessment M2e-specific IgG.All strains all present the M2e-specific IgG of significant quantity, and the immunogenicity of expression Pam3Cys.M2e does not rely on specific MHC (Figure 57).

Immunogenicity in rabbit

Carry out research purpose and be, assessment Pam3Cys.M2e and STF2.4xM2e are in the immunogenicity of second species (rabbit).In first research, be in the 0th, 21 and 42 day, rabbit (3 male and 3 female/group) is caused with the Pam3Cys.M2e of 500,150,50,15 or 5 μ g (i.m.) exempt from.Organize in contrast, and additional set acceptance blending buffer F111 (10mM Tris, the 10mM histidine, 75mM NaCl, 5% sucrose, 0.02% poly-sorbose acid esters-80,0.1mM EDTA, 0.5% ethanol, 20mg/mL hydroxyl third-beta-schardinger dextrin-, pH7.2).Obtained peripheral blood on the 7th day in short 2 backs that rise, and resist-the M2e antibody titer by the ELISA assessment.Be shown in the presentation of results of Figure 58, the indivedual rabbit antibody titers under the 1:125 serum dilution.Elementary/short Pam3Cys.M2e amount that rises vaccination that data suggest is used for, and between the antibody titer degree that is reached dose-response relationship is arranged.

In second research, be rabbit (3 male and 3 female/group) to be caused with the STF2.4xM2e of 500,150,50,15 or 5 μ g (i.m.) exempt from.Organize in contrast, an additional set is only accepted saline solution.Exempt to obtain in back the 14th day peripheral blood in causing, and resist-the M2e antibody titer by the ELISA assessment.Especially, exempt from the animal through causing, before after the initiation the 14th day, can detect tangible M2e-specific IgG reaction (Figure 59) in all.Described result points out that STF2.4xM2e causes quick and consistent immunoreation in rabbit.

Excite the effect of model in mice

Use quite characterization mice adaptation influenza A virus strain in mice, A/ Puerto Rico/8/34 (H1N1) assesses the effect of Pam3Cys.M2e and STF2.4xM2e as challenge virus.With every group of ten mices in the 0th and 14 day, there is blending buffer F111 (■) through s.c. with the Pam3Cys.M2e of 30 μ g, there is exclusive buffer F120 (10mM Tris in the Pam3Cys.M2e of 30 μ g, the 10mM histidine, 10% sucrose, 0.02% poly-sorbose acid esters-80,0.1mM EDTA, 0.5% ethanol, 0.075% docusate sodium, pH7.2) (▲), 30 μ g Pam3Cys.M2e exist buffer F119 (10mM Tris, 10mM histidine, 75mM NaCl, 5% sucrose, 0.02% poly-sorbose acid esters-80,0.1mM EDTA, 0.5% ethanol, 0.1% docusate sodium, pH 7.2), 30 μ g of STF2.4xM2e exist buffer F105 (10mMTris, 10mM histidine, 75mM NaCl, 5% sucrose, 0.02% poly-sorbose acid esters-80,0.1mMEDTA, 0.5% ethanol, pH 7.2), 3 μ g STF2.4xM2e exist buffer F105 (10mM Tris, 10mM histidine, 75mM NaCl, 5% sucrose, 0.02% poly-sorbose acid esters-80,0.1mM EDTA, 0.5% ethanol, pH 7.2) (●) or 0.3 μ g STF2.4xM2e exist buffer F105 (√) to cause to exempt from.Comprise that a winding is subjected to independent PBS as negative control group (zero), and one group after sublethal dose excites with virus, have to convalescent period of PR/8 group as positive controls ().In the 28th day, with LD ₉₀PR/8/34 excite reserve to excite.Reach 14 days in exciting back investigation body weight to run off and survive.

The animal of convalescent period group is successfully removed early stage nonlethal PR8 and infects, and proves follow-up virus excited to have 100% protective effect.Accept animal of independent PBS buffering, began to take on morbit forms, 80% fatality rate took place in the 10th day before, and through the animal that the Pam3Cys.M2e of 30 μ g exists F111 to cause to exempt from, prove its survival rate increase (have 50% survive in this excite) in the 7th and 8 day.There is the animal of the Pam3Cys.M2e of buffer F119 in acceptance, begins to take on morbit forms in the 8th and 9 day, and the survival of 80% mice is arranged.There is buffer 120 (10mM Tris in acceptance, the 10mM histidine, 10% sucrose, 0.02% poly-sorbose acid esters-80,0.1mM EDTA, 0.5% ethanol, the proteinic animal of Pam3Cys.M2e pH7.2) or STF2.4xM2e, present the gentleest lysis, have 90 to 100% mices to survive in this in these groups and excite.These results prove that Pam3Cys.M2e and STF2.4xM2e can grant and in vivo resist the protective immunity that excites with influenza A.

Discuss

Salmonella typhimurium flagellin (FljB) is the part of TLR5.Repeat the recombinant protein formed by total length flijB (STF2) through merging to four series connection of M2e in expression in escherichia coli, and purified to 95% purity has low endotoxin content.In report cell line, this protein (STF2.4xM2e) causes IL8 in TLR5-dependence mode and makes.STF2.4xM2e diluent through scope from 0.25 to 25 μ g (allocate in buffer F105, do not contain known adjuvant or supporting agent) causes the mice of exempting from, and produces violent antibody response.In animals received wherein few as 5 μ g in single dose after in the proteinic rabbit immunogenicity research of serum conversion, further prove the effect of recombinant protein.In using influenza A/ Puerto Rico/8/34 mice to excite in the model, prove the effect of PAMP fusion rotein as challenge virus.The infrequent menstruation every dosage of 0.3 μ g protein according to appointment causes the morbid state that the mice of exempting from presents gentleness, has 100% mice to survive in this and excites.

The acidylate amino terminal of synthesis type three palmityl peptides simulation lipid bacterioprotein, and be effective TLR2 activator.In these researchs, be to use the standard solid phase peptide chemosynthesis to get, comprise the cysteine residues of binding functional domain (M2e) to influenza A M2 born of the same parents and three palmityl peptides of aminoterminal tri-fatty chain.This peptide (Pam3Cys.M2e) relies on mode with TLR2-, causes TNF α and make in report cell line.Be used to cause when exempting from mice under no adjuvant exists, Pam3Cys.M2e produces than M2e more effective antibody response when mixing with free state Pam3CSK-4.Pam3Cys.M2e also has immunogenicity in being found in rabbit, wherein observe to be used to cause the Pam3Cys.M2e amount of exempting from, and between the antibody titer that is reached dose-response relationship is arranged.In using influenza A/ Puerto Rico/8/34 mice as challenge virus to excite in the model, there is the effect of the Pam3Cys.M2e peptide in many different concoctions in assessment.The Pam3Cys.M2e that allocates in F119 and F120 presents the gentleest morbid state, has an appointment 80 to survive in this to about 100% mice and excite.

Embodiment 20

Materials and methods

Pcr amplification and dna primer

All pcr amplification effects are to use, and (La Jolla, Pfu Ultra HotstartPCR Master Mix (catalog number 600630) CA) finishes according to manufacturer's recommendation available from Shi Tajin.Dna primer is available from sigma Genosys, and through being described in down.

STF28BGF-1：

CTCGGGAGATCTGCACAAGTAATCAACACTAACAGTCT(SEQ ID NO：644)

STF28MCR-1：CCATGGGCTAGCAGGATCCACCGGCGCTCCCTGCACGTTCA(SEQ ID NO：645)

STF28MCF-2：GGAGCGCCGGTGGATCCTGCTAGCCCATGGACCGAAAACCCG(SEQ IDNO：646)

STF28ECR-2：

TCTGCAGAATTCACGTAACAGAGACAGCACGTTCTGCGGGACGTCCCGCAGAACGTGCTGTCTCTGTTA

CGTGAATTCTGCAGA(SEQ ID NO：647)

pET24AR：5 TCCGGCGTAGAGGATCGAGA(SEQ ID NO：648)

STF2-E3R3：

CAATTGACCTTCAAGCTTCGAATTGCCCTTACGTAACAGAGACAGCACGTTCTG(SEQID NO：649)

AX-E3F3：

AAGCTTGAAGGTCAATTGGAATTCCCTAGGACTAGTATGGAAAAATTGCAGTTGAAG(SEQ ID NO：650)

pET24AF：GCTTAATGCGCCGCTACAGG(SEQ ID NO：651)

5’WNE28：GCGGCCGCTCATGGAAAAATTGCAGTTGAAGGGAACAACC(SEQ IDNO：652)

3’WNE28：CCGCGGTTTGCCAATGCTGCTTCCAGACTTGT(SEQ ID NO：653)

NdeI-STF2：

CCGGCATGCCATATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGC(SEQ ID NO：654)

BlpI-EdIII：GCATGCTCAGCTTATTAAGGGTTTGCCAATGCTGCTTCCCAGACTTGTG(SEQ ID NO：656)

JE EIII primer: TACGTGAATTCAGCAGATATCCAGCAC (SEQ ID NO:53)

The clone of pET/STF2 Δ .EIII

The total length flagellin fljb of salmonella typhimurium (flagellin phase 2) (also being called ＂ STF2 ＂ in this paper) is by the 1.5kb coded by said gene.The hypervariable region disappearance of the amino acid/11 70 to 415 by will crossing over SEQ ID NO:604, and produce the truncate version (STF2 Δ, SEQ ID NO:606 is by SEQID NO:607 coding) of STF2.Help TLR5 to summon the effect of necessary NH2 and COOH end sequence with one section through design the disappearance regional replacement, lack flexible peptide connexon (GAPVDPASPW, SEQ IDNO:659).For producing this construct, use two step PCR then.In first reaction, be to use STF2.OVA (SEQ ID NO:755 encoding amino acid sequence SEQ ID NO:756) as dna profiling, it is right as primer to reach STF28BGF-1 and STF28MCR-1.In the reaction that separates, be with same DNA template and primer STF28MCF-2 and STF28ECR-2 combination.

Described pcr amplification reaction produces about 500bp and about 270bp fragment respectively.These PCR products are combined in use STF28BGF-1 and STF28ECR-2 to react as the final PCR of primer.With reaction from then on increase DNA product (about 0.77kb), decompose with BglII and EcoRI restriction endonuclease, and be engaged in advance through BglII and EcoRI digest the pMTBiP/V5-HisB that reaches with alkaline phosphatase treatment (Yin Weituo King Company, Ka Sibeide, CA) in.Get equal portions engage mixture be used to transform the TOP10 cell (Yin Weituo King Company, Ka Sibeide, CA).Use the carrier specificity primer, pMTFOR (methionine promoter) (CATCTCAGTGCAACTAAA, SEQ ID NO:759) with BGHREV (bovine growth hormone is gathered A) (TAGAAGGCACAGTCGAGG, SEQ ID NO:760) carry out the PCR screening, and identify several positive clone strains.The total positives clone strain is further analyzed by the estriction map analysis, and confirmed by the DNA sequencing.The construct pMT/STF2 Δ that is become is used to produce pMT/STF2 Δ .EIII+.

The functional domain III of the west nile virus envelope protein of pET/STF2 Δ .EIII+ (SEQ ID NOS:673,674) is derived from fruit bat expression plasmid pMT/STF2.E.This plasmid contains total length STF2 (amino acid/11-506, SEQ ID NO:604) and merges to west nile virus envelope protein (amino acid/11-406, SEQ ID NO:642).PMT/STF2.E (SEQ ID NO:761) clone strain AX-1 is used as dna profiling, and 5 ' WNE28 (SEQ ID NO:652) and 3 ' WNE28 (SEQ ID NO:653) as primer to carrying out pcr amplification.For helping restriction analysis and follow-up clone's step, new NotI of this 5 ' primer coding cuts the position, and (MA), and 3 ' primer contains unique SacII and cuts the position for New England Biolab, Bei Fuli.Will be through the EIII+DNA fragment (345bp of amplification; The aminoacid 292-406 of its coding of SEQ ID NO:781 SEQ ID NO:642), time cloning go into pCR-Blunt II-TOPO cloning vehicle (Yin Weituo King Company, Ka Sibeide, CA) in, and produce plasmid TOPOEIII.Then pass through to use T4DNA polymerase passivation SacII and SpeI restriction site, and termination codon is imported the downstream of EIII+ sequence.

For producing pMT/STF2 Δ .EIII+ (SEQ ID NOS:673,674), use NotI and BamHI restriction site are isolated the EIII+ fragment from TOPOEIII+, and engage into NotI and SacII restriction site in the pMT/STF2 Δ.Before engaging, the segmental BamHI of EIII+DNA is cut the position cut the position, with the passivation of T4DNA polymerase with the SacII of pMTSTF2 Δ.By using primer NdeI-STF2 and BlpI-EdIII to carry out pcr amplification, STF2 Δ .EIII+ sequence (SEQ ID NOS:673,674) is isolated from pMT/STF2 Δ .EIII+.For producing pET/STF2 Δ .EIII+ (SEQ ID NO:674), the PCR product is decomposed with NdeI and BlpI, and be engaged in advance in the pET24a plasmid of NdeI and BlpI digestion.To engage mixture be converted into the Mach-1 cell (Yin Weituo King Company, Ka Sibeide, CA) in, and cell grown on the LB that replenishes with 50 μ g/ml kanamycin.Get several clone strains by the estriction map Analysis and Identification, and confirm by the DNA sequencing.

The clone of pET/STF2.EIII+

The EIII+ sequence of pET/STF2.EIII+ (SEQ ID NOS:657,658) is derived from pETSTF2.E (SEQ ID NOS:761,762).This colibacillus expression plasmid contains total length STF2 (amino acid/11-506, SEQ ID NO:604), through merging to west nile virus envelope protein (the aminoacid 292-406 of SEQ ID NO:642, it is SEQ ID NO:610).In two independent PCR reactions, be to use pET/STF2.E as dna profiling.Primer pET24AR:5 (SEQ ID NO:648) and STF2-E3R3:(SEQ ID NO:649 are used in a reaction), and another uses AX-E3F3 (SEQ ID NO:650) and pET24AF (SEQ ID NO:651).These PCR reaction produces a 1.5kb fragment of being made up of total length STF2, and comprises the EIII functional domain and add the 340bp fragment that extends into the additional amino acid among the envelope protein functional domain I.With each equal portions combination of these PCR reaction, and with this two product as template, be used for carrying out the PCR reaction with outside primer pET24AR (SEQ ID NO:648) and pET24AF (SEQ ID NO:651).This causes producing about 1.8kb dna fragmentation, and it merges to STF2 EIII+ sequence (SEQ ID NO:781 is the nucleotide sequence of the aminoacid 292-406 of coding SEQ ID NO:642, and it is SEQ ID NO:610).The PCR product is decomposed with NdeI and BlpI, and is engaged in advance through compatible enzymic digestion and go-the pET24a carrier of phosphorylation in.To engage mixture as preceding about as described in the pET/STF2 Δ .EIII+, be converted into the Mach-1 cell (Yin Weituo King Company, Ka Sibeide, CA) in.Get several clone strains by the estriction map Analysis and Identification, and confirm by the DNA sequencing.

The clone of pET/STF2 Δ .JEIII+

Japanese encephalitis virus (the JEV) (part of the envelope protein of strain SA-14-14-2 (Jai, people such as L., Chin Med J (Eng) 116:941-943 (2003)); The JEV vaccine that is used at present by functional domain III coding, is (to cover Luo Gongyuan, CA) customized synthesizing by DNA2.0 Inc..Use the NotI and the Blp I that are positioned at insert two flanks to cut the position this partial function territory III, cut out from pJ2:G01510.The DNA insert with gel separation, and is cloned into via compatible end, disengages among the pET24A/STF2 Δ .EIII+ (SEQ ID NOS:673,674) of west nile virus EIII+ insert through suitable enzymic digestion in advance.Carrier that then will disappearance is with gel-purified and be engaged to the packing of a JE EIII+.To engage mixture is used to transform the TOP10 cell (Yin Weituo King Company, Ka Sibeide CA), and grows in cell on the LB that replenishes with 50 μ g/ml kanamycin.Get several clone strains by the estriction map Analysis and Identification, and confirm by the DNA sequencing.

The construct that is become, pET24A/STF2 Δ .JEIII (SEQ ID NOS:608,609) be BLR (DE3) strain (Nova gold), and use the expression in several clone strains of the blue dyeing monitoring of Kao Maxi, it is confirmed by the western blotting that uses anti--flagellin antibody.Use pET24A/STF2 Δ .JEIII+ as dna profiling, and JE EIII+ oligonucleotide is as primer (SEQ ID NO:656), use the QuikChange fixed point to cause and become test kit (Shi Tajin, La Qiaola, CA) according to the explanation of manufacturer, will between and peptide connexon zone in cysteine residues change to serine residue.Confirm clone strain by sequencing, and as preceding about its expression of analysis as described in the pET/STF2 Δ .EIII+.

When the cysteine residues in the peptide connexon zone changes to serine residue, in this fused protein, include ＂ s ＂ in, name this fused protein.For example, " STF2 Δ .EIII+ " comprises the cysteine residues (SEQ ID NO:674) that is arranged in peptide connexon zone, and " STF2 Δ .EIIIs+ " comprises the serine residue (SEQ ID NO:675) that is substituted the cysteine residues that is arranged in peptide connexon zone.

The EIII functional domain of cloning each dengue virus is through merging to the C-end of flagellin (STF2 Δ)

At first, it is difficult obtaining the tool bioactive substance from the fusions of west nile virus complete packet memebrane protein, may be owing to there are most cysteine residues (12 cysteine) (referring to, SEQ ID NO:642) in envelope protein.Yet when time cloning was carried out in the zone of this protein functional domain III (EIII) that will encode, this fused protein is abundant the expression in escherichia coli, and effective in the mice camber.Though between the different DEN viruses of 4 strains (Den1, Den2, Den3, Den4, SEQ ID NOS:763-770) whole sequence dissimilarity is arranged, the three dimensional structure in the envelope protein functional domain III between banzi virus is similar.This functional domain in DEN and other banzi virus, important/advantage neutrality epitope that the most type-specificity of encoding is adjacent.The functional domain III of dengue virus (Den1, Den2, Den3 and Den4), be expressed in the antibacterial and shown and had immunogenicity, can in laboratory animal, induce neutrality antibody (west is covered, people such as M., Am.J.Trop.Med Hyg 65:159 (2001)).Corresponding to the residue about 295 of the different DEN viruses of the four strains functional domain III to about 399 (correct count is to decide on specific DEN virus, for example SEQ ID NOS:763,765,767,769), the codon optimization is used for escherichia coli expression.Synthetic gene is used pcr amplification, and the NotI that time cloning is gone into carrier pET/STF2 Δ cuts in the position, produces pET/STF2 Δ .DEN1EIII, pET/STF2 Δ .DEN2EIII, pET/STF2 Δ .DEN3EIII and pET/STF2 Δ .DEN4EIII (SEQ ID NOS:683,685,687 and 689).

The escherichia coli of STF2.EIII+, STF2 Δ .EIII+, STF2 Δ .EIIIs+ and STF2 Δ .JEIII+ are made

To have pETSTF2.EIII+ (SEQ ID NOS:657,658), pETSTF2 Δ .EIII+ (SEQ IDNOS:673,674), pETSTF2 Δ .EIIIs+ (SEQ ID NOS:675,676) or pETSTF2 Δ .JEIII+SEQ ID NOS:608,609) cell culture (6L) of BLR (DE3) pLysS grows in the LB culture medium that contains 15 μ g/ml kanamycin, 12.5 μ g/ml tetracyclines and 24 μ g/ml chloromycetin.In OD ₆₀₀Be about 0.6 time, express in 37 ℃ of down about 3h induced proteins with 1mM IPTG.After inducing, cell is collected by centrifugal (7000rpm x 7 minutes in Sorvall RC5C centrifuge), and resuspending is in 2X PBS, 1% glycerol, 1 μ g/ml DNAseI, 1mM PMSF, protease inhibitor cocktail and 1mg/ml lysozyme.Suspension is made cytolysis by little liquefier, and with lysate centrifugal (in Beckman Optima L supercentrifuge, in 45,000g next hour), so that soluble part separates with inclusion body.Under these growths and breeding condition, STF2.EIII+ expresses and is soluble protein, and STF2 Δ .EIII+ (SEQ ID NOS:673,674), STF2 Δ .EIIIs+ (SEQ ID NOS:675,676) and STF2 Δ .JEIII+ (SEQ ID NOS:608,609) form inclusion body.

The purification of STF2.EIII+

The cell lysates that will contain solubility STF2.EIII+ (SEQ ID NOS:657,658), in 0.5M NaCl exist down application of sample to agarose Q resin (the Ai Moxun bioscience, skin SIKA tower prestige, NJ) on, to reduce DNA, endotoxin and other impurity.Merchantable thing collected and by doubly diluting adjustment electric conductivity with buffer A (buffer A: 100mM Tris-Cl, pH 8.0) 10-.With diluted material again application of sample to Q agarose tubing string, and the linear gradient elution of conjugated protein with 20% to 60% buffer B (pH 8.0 for buffer B: 100mMTris-Cl, 1M NaCl) gone out.The stream part that to contain STF2.EIII+ is compiled, and further with Superdex-200 gel (SD200) filtration chromatography art, there is processing down in the Na-dexycholate, to remove residual endotoxin (execution buffer: 1% Na-dexycholate, 100mM NaCl, 100mM Tris-HCl, 1% glycerol, pH 8.0).In analysing postoperative, eluting is gone out the direct application of sample of lipoprotein to the Q agarose, and fully wash the removal cleaning agent with buffer A by the SD200 filtering layer.Conjugated protein is gone out with the linear gradient elution of 20% to 60% buffer B once more.In a preparation (batch part 057), be with this step replace with, (cleaning agent IL) removes program for Pierre's Si biotechnology, Luo Kefo to use extraction-D cleaning agent removal gel.Purified protein is dialysed to the buffer that contains 50mM Tris, 100mM NaCl and 1% glycerol, and store in-80 ℃.

The purification of STF2 Δ .EIII+

Inclusion body is collected by low-speed centrifugal (7000rpm x 7 minutes in Sorvall RC5C centrifuge), and to contain 8M carbamide, 100mM Tris-HCl, 5mM EDTA, the buffer dissolving of pH8.0.The urea concentration of solubilising protein is adjusted to 1M, and with sample application to the Q agarose.The linear gradient elution of conjugated protein with from 0% to 100% buffer B gone out.(buffer A: 100mM Tris-HCl, 5mM EDTA, 1M carbamide, pH8.0.Buffer B: 100mM Tris-Cl, 1M NaCl, pH8.0).Owing to form protein aggregate behind eluting, the urea concentration of satisfying Q agarose material is adjusted to 8M.Protein is further purified by the gel permeation chromatography art of using SD200.With tubing string with 100mMTris-HCl, pH8.0,100mM NaCl, 1% glycerol, 8M carbamide add 1% Na-cholate pre-balance.The protein that eluting is gone out uses sucrose Q to carry out the 2nd IEX chromatographic step, to remove the Na-cholate.The linear gradient elution of conjugated protein with from 20% to 60% buffer B gone out.(buffer A: 100mMTris-Cl, pH8.0,8M carbamide, 5mM EDTA.Buffer B: 100mM Tris-Cl, 1M NaCl, pH8.0).By the gel permeation chromatography art (execution buffer: 100mM Tris-HCl, pH8.0,8M carbamide, 100mM NaCl and 1% glycerol) of using SD200, finish proteinic last retouching.Reducing agent is added to SD200 stream part (a 2.5mM DTT), and with protein by the carbamide of concentration is decrescence progressively dialysed and is folding again.Urea concentration is to containing 100mM Tris-HCl, pH8.0,100mM NaCl, 1% glycerol and 6M, 4M, 2M or do not have the buffer of carbamide, and lower in regular turn.

The trimerical folding again and purification of STF2 Δ .EIII+

STF2 Δ .EIII+ (the SEQ ID NOS:673 of carbamide stabilization inclusion body must hang oneself, 674) by simply dialysis as the aforementioned, folding effectively again and form trimer, be that the basis is estimated as trimer (STF Δ .EIII fusion rotein 3 times) with the molecular weight among the SDS-PAGE.After dialysis, endotoxin is removed by extracting for many times with Triton X-114.By S200 size exclusion tomography, with trimer from monomer and aggregation purification and isolate.End product is gone up to move in SDS-PAGE and is presented single band, and it is about 130kDa that its demonstration has molecular weight.

The monomeric folding again and purification of STF2 Δ .EIII+

Folding again by what use rapid dilution to carry out, consistent and make STF2 Δ .EIII+ (SEQID NOS:673 effectively, 674) monomeric form, it prevents that indivedual STF2 Δ .EIII+ fused proteins from can not interact with each other and form polymer, for example trimer (as described above).The 8M carbamide that will exist 4M carbamide to increase to from the STF2 Δ .EIII+ of inclusion body stabilization not contain Reducing agent.Then with protein under room temperature rapid dilution in Tris/NaCl/ glycerol buffer, pH8.0, reaching middle urea concentration to about 0.1mg/ml is 0.1M.By S200 size exclusion tomography, with monomer from the aggregation purification and isolate.End product is gone up to move in SDS-PAGE and is presented single band, and it is about 43kDa that its demonstration has molecular weight.

(serine of the peptide connexon between between STF2 Δ and EIII+ replaces STF2 Δ .EIIIs+, SEQ ID NO:675) replacement

Use the similar folding monomeric rapid dilution method of STF2 Δ .EIII+ that is used for again, the STF2 Δ .EIIIs+ of the stabilization inclusion body of must hanging oneself (SEQ ID NOS:675,676) is folding again.To through folding again protein capture on the butyl-agarose tubing string and eluting, remove most of contaminated with endotoxins simultaneously.To concentrate from the eluate of butyl-agarose purification, and by 4 Triton X-114 extraction cycle, so that endotoxin concns is reduced to pact＜0.1EU/ μ g by before the final purification step of SD200 gel filtration.Finally compile product and present single band in SDS-PAGE is upward mobile, it is about 43kDa that its demonstration has molecular weight, and contains the Triton X-114 (about 0.000015%) of trace.

The purification of STF2 Δ .JEIII+ (SEO ID NOS:608,609)

Protein is to separate from inclusion body under the degeneration condition.Inclusion body with cleaning agent cleaning and stable in 8M carbamide, is caused the proteinic special purification of target.About endotoxin removal, be with the protein application of sample on the sucrose S cation exchange tubing string of low pH value (about 3.5), and with salt gradient (0 to about 1M NaCl) eluting.Use as carrying out for the described rapid dilution of STF2 Δ .EIII+ monomer again protein folding.Then protein is concentrated, and use S200 size exclusion tomography, monomer is isolated from aggregation.Purified material is gone up to move in SDS-PAGE and is presented single band, and it is about 43kDa that its demonstration has molecular weight, and contains the endotoxin (about 0.03EU/ug) of acceptable degree.

The stream of fusion rotein adds formula (Fed Batch) manufacturing

In aerobe reactor, use stream to add the formula processing procedure and make STF2 Δ .EIIIs+.Place three control loops, by adding acid (2N HCl) or alkali (3N NH ₄OH) regulation and control pH value, by heating (heat and coat blanket) or cooling (time circulation air ring) regulating and controlling temperature, and by compressed air stream (people is for controlling), stirring (mixing velocity) and O ₂Stream (timing controlled) polyphone regulation and control dissolved oxygen amount.To have STF2 Δ .EIIIs+ [BLR (DE3) pLysS is cell adapted and be stored in MRSF culture medium (referring to aforementioned), and is frozen in 25% glycerol.Cell by in the MRSF culture medium that 1mL is stored cell and add to 1L, and was stirred about 15.5 to 16.5 hours down in about 37 ℃, and in the bioreactor amplification culture.The cell that derives from amplification procedure with about 1:10 ratio, under about 37 ℃ and about 0.5vvm air flow, is added to MRSF or MRBR synthetic medium.

Carry out down this processing procedure with batch-mode in 37 ℃, cause compressed air stream to be about 1.5vvm, and stirring reaches (about 6 hours) till the maximum, when temperature is reduced between about 25 ℃ and about 33 ℃ up to the cell oxygen expenditure.Can be in carrying out before culture induces, or in after about 1 to about 1/2 hour beginning feeding.Can make charging rate keep constant, or adjust it based on processing procedure parameter (dissolved oxygen amount, concentration of glucose).When a batch part glucose exhausts, culture is induced with IPTG.Culture was kept minimum about 2 hours, and be about most 20 hours.

The MRBR culture medium Trace meter solution 1000x

Form G/L Form G/L

Glucose 20 EDTA, disodium 5

KH ₂PO ₄ 2.2 FeSO ₄(7H ₂O) 10

(NH ₄) ₂SO ₄ 4.5 ZnSO ₄(7H ₂O) 2

Citric Acid 1.0 MnSO ₄(H ₂O) 2

MgSO ₄(7H ₂0) 1.0 CoCl ₂(6H ₂O) 0.2

CaCl ₂ 0.04 CuSO ₄(5H ₂O) 0.1

Trace meter 1ml Na ₂MoO ₄(2H ₂O) 0.2

Thiamine HCl 0.01 H ₃BO ₃0.1

Defoamer 0.05

The MRSF culture medium The feeding culture medium

Form G/L Form G/L

Glucose 10 (20 in bioreactor) NaCl 0.5

KH ₂PO ₄ 7.8 FeSO ₄(7H ₂O) 2

(NH ₄) ₂SO4 2.33 CaCl ₂ 3.5

Citric acid 1.0 MgSO ₄(7H ₂O) 12

MgSO ₄(7H ₂O) 1.0 thiamine HCl 1

CaCl ₂0.04 trace meter 1ml

Trace meter 1ml glucose 100

Thiamine HCl 0.01

Kanamycin 0.0075 (only being used to shake bottle)

STF2 Δ .EIIIs+ is inclusion body to make.When collecting by centrifugal (Beckman Avanti J-20 XP, JLA 8.1000 turns, 10kxg in about 4 ℃ about 20 minutes down) cell is separated with conditioned medium, and be suspended in isopyknic 50mM Tris, 100mM NaCl, 1mM EDTA is among the pH 8.0.Repeated centrifugation under the same terms, with the cell resuspending in the same buffer of minimum volume.With suspension in 10,000 times by homogenizer (APV-1000) at least twice.

Solid can be separated, and will be by the dissolving wherein of following three kinds of methods; Centrifugal, filter or fluid bed chromatography art.

Method 1

With solid by centrifugal (Beckman Avanti J-20 XP, JA 20 turns, 20kxg in about 4 ℃ about 20 minutes down) separate, and resuspending is in 50mM tris, 1m NaCl, 1mM EDTA, 1% glycerol, 0.5%Triton X-100 is among the pH 8.0.In cumulative speed and under the time (reaching about 20 minutes for about 40kxg), repeat this program 6 times (total) at the most to maximum.After last precipitation reclaimed, will precipitate resuspending in 50mM Tris, 0.1M NaCl, 1mM EDTA, among the pH 8.0, and by centrifugal (Beckman AvantiJ-20 XP, JA 20 turns, 40kxg was in about 4 ℃ times about 20 minutes) clarificationization.To precipitate resuspending and be dissolved in 50mM Tris, 0.1M NaCl, 1mM EDTA, 4M carbamide is among the pH 8.0.With soluble thing by centrifugal (Beckman Avanti J-20XP, JA 20 turns, 40kxg in about 4 ℃ about 50 minutes down) remove, keep supernatant and be used for further processing.

After repeatedly cleaning as the aforementioned, also STF2 Δ .EIIIs can be dissolved in 50mM acetate, 10mMNaCl, 8M carbamide, among the pH about 4.1 to about 5.3, and by centrifugal (Beckman Avanti J-20 XP, JA 20 turns, and 40kxg descended about 20 minutes in about 4 ℃) clarificationization.

Method 2

After homogenizing, lysate is trapped in the body feeding (body feed), and STF2 Δ .EIIIs+ is extracted with the buffer that contains carbamide.The body feeding be through design in order to particle trapping in, be arranged in the filter auxiliary agent of the filter cake on the bottom filter.This body feeding (CelPure 65 of Advanced Minerals company (AdvancedMinerals Corporation)) is for having high surface and the low penetration kieselguhr (silica powder) of (keeping＜0.2 μ m granule).Filter auxiliary agent and lysate are pre-mixed, and pass through bottom filter (Ertel 703), set up and contain body feeding and the particulate filter cake of lysate with the group Pu.When granule Shen was amassed on filter bed, suspension produced the bottom filter.Carry out 50mM Tris, 100mM NaCl pH 8.0 flushings are to remove soluble protein and nucleic acid.Follow with 50mM Tris, 100mM NaCl, 4M urea, pH8 washes, and stable reaching shifted out STF2 Δ .EIIIs for further processing from the body feeding.

Method 3

With cell at first resuspending in buffer after, in the buffer that contains sodium chloride and carbamide, pH about 6 is to about 8 with they's resuspending, and homogenizes.In Streamline CST fluid bed tubing string (GE health-care (GE Healthcare)), wherein STF2 Δ .EIIIs+ is bonded to this resin and microgranule circulates out with the lysate application of sample.STF2 Δ .EIIIs+ can be under low-salt conditions, in the pH value that is higher than application of sample pH, in having or not having cleaning agent for example Triton X-100 or poly-sorbose acid esters-80 exist down that eluting goes out.

SDS-PAGE

Protein (about typically 5 μ g) is diluted in or does not contain in the SDS-PAGE sample buffer of beta-mercaptoethanol.Sample is boiled 5 minutes, and application of sample is to the 4-20% sds page.Through the electrophoresis postoperative, gel is dyeed to present protein band so that Kao Maxi is blue.

Endotoxin analysis

(BioWhittaker #50-648U, Walker's paddy MD), according to the explanation that manufacturer is used for the micro plate method, are measured endotoxin concns to the LAL test kit to use QCL-1000 quantitatively to develop the color.

Protein analysis

By with 96-hole form, use MicroBCA protein analysis reagent test kit (Pierre Si biotechnology, Luo Kefu, IL) the mensuration protein concentration of BSA as reference material.

The TLR5 biological activity is analyzed

HEK293 cell (ATCC, catalogue No.CRL-1573 Manassas, Virginia) is expressed TLR5 inherently, and in the reaction to the TLR5 citation, secretes several soluble factors (comprising IL-8).With cell inoculation (about 50,000 cells/well) in the micro plate of 96-hole, fused protein adding and cultivation are spent the night.Next day, collect conditioned medium, be transferred to clean 96-hole micro plate, and freezing down in-20 ℃.After waiting to thaw, in the antibody that uses Anti-Human's class-IL-8 to cooperate to (Pierre Si, #M801E and #M802B, Luo Kefu, sandwich ELISA Il) exists according to the IL-8 in the explanation analysis condition culture medium of manufacturer.(skin SIKA tower prestige NJ) is measured optical density (OD) for FARCyte, Ai Moxun bioscience to use the micro plate spectrophotometer.

Plaque reduces neutralization test (PRNT)

According to people such as kings, J.Immunol.167:5273-5277 (2001) carries out PRNT.Letter speech, serum sample is deactivated by cultivating in 56 ℃ of water-baths about 30min heat, and serial dilution is in the PBS that contains 5% gelatin from 1/10 to 1/2560.West nile virus is diluted among the PBS that contains 5% gelatin, so that final concentration is about 100PFU/ hole.Virus and about 75 μ l serum are mixed in the 96-orifice plate about 1h under about 37 ℃.With the serum-virus mixture of equal portions cultivate in, grow in being paved with on the Vero cell monolayer of six-hole tissue culturing plate.Cell is cultivated 1h under about 37 ℃, and with plate vibration in U.S. 15 minutes.The agar cover layer is added.This cover layer be by with equal-volume by the solution that 100ml 2x MEM (life science and technology (LifeTechnologies)) forms, mix with aseptic 2% agar and be prepared into.Two solution are placed 40 ℃ of water-baths 1 hour, add cover layer afterwards.With cell culture under 37 ℃, in 5% CO through humidity ₂Cultivated 4 days in the-air mixture.Second cover layer that will have extra 4% dimethyl diaminophenazine chloride in the 5th day adds.The viral plaque of counting after about 12 hours.

The antigenicity of STF2 Δ-fusion rotein

Elisa plate (96-hole) under 4 ℃, is applied and spends the night with the serial dilutions (100 μ l/ hole) of purified STF2 Δ-fused protein (SEQ ID NOS:761,762,657,658,673,674) in PBS (about 2 μ g/ml).With the analysis dilution buffer liquid (ADB of plate with 200 μ l/ holes; BD Fa Minggen) under room temperature, blockaded one hour.Plate is cleaned in PBS-Tween 3 times, cultivate with flagellin or the reactive antibody of E functional domain tool then construct.Use mAb 6H11 (Intotek) to detect the expression of flagellin, and use one group available from Bioreliance (Lip river Ke Gu, mAb MD) (5C5,7H2,5H10,3A3 and 3D9), the antigenicity (Beasley of monitoring WNV-E, D.W. wait the people, J.Virol.76:13097-13100 (2002)).Antibody (the about 100 μ l/ holes) cultivation under 4 ℃ that is diluted in ADB is spent the night.Plate is cleaned 3 times with PBS-T.With the HRP-labelled goat that is diluted in ADB anti--(Jackson's immunochemistry, Xi Geluofu PA) add (100 μ l/ hole) to mouse IgG antibody, and plate was cultivated under room temperature 1 hour.Plate is cleaned 3 times with PBS-T.(Pierre's Si biotechnology, Luo Kefu IL), and behind the monitoring color colour generation, measure A450 to TMB to be added (3,3 ', 5,5 '-tetramethyl benzidine) Ultra substrate on Tecan Farcyte micro plate spectrophotometer.

Causing of mice exempted from

C3H/HeN mice (10 every group) in the 0th, 14 and 28 day, is caused and exempts from through intraperitoneal or subcutaneous fusion rotein or synthetic peptide with specified concentration.In the 21st day and 35 days, will exempt from animal by puncture blood collecting behind the eye socket through causing.Do not condense and the centrifugal serum of gathering by will there being the heparinemia fluid samples.In the 35th day, the WNV Strain 2741 (king, people such as T., J.Immunol 167:5273-5277 (2001)) of mice with fatal dose excited.In exciting back its survival of monitoring to reach 21 days.

Serum antibody is measured

Measure Xi Niluo envelope protein specific IgG concentration by ELISA.With elisa plate (96-hole) under 4 ℃, be dissolved in western Buddhist nun sieve E albumen mAb 5C5,7H2,5H10,3A3 and the 3D9 (Beasley that PBS concentration is 2 μ g/ml with 100 μ l/ holes, D.W. wait the people, J.Virol.76:13097-13100 (2002)) (Bioreliance, Lip river Ke Gu MD) applies and spends the night.With the analysis dilution buffer liquid (ADB of plate with 200 μ l/ holes; BD Fa Minggen, Santiago CA) under room temperature, blockaded one hour.Plate is cleaned in PBS-T 3 times.Serum is added (100 μ l/ hole) in the diluent of ADB, and plate cultivation under 4 ℃ is spent the night.Plate is cleaned 3 times with PBS-T.With the HRP-labelled goat that is diluted in ADB anti--(Jackson's immunochemistry, Xi Geluofu PA) add (100 μ l/ hole) to mouse IgG antibody, and plate was cultivated under room temperature 1 hour.Plate is cleaned 3 times with PBS-T.(Pierre's Si biotechnology, Luo Kefu IL), and behind the monitoring color colour generation, measure A to TMB to be added (3,3 ', 5,5 '-tetramethyl benzidine Pierre Si) Ultra substrate on Tecan Farcyte micro plate spectrophotometer ₄₅₀

The synthetic manufacturing of Pam3Cys.WNV001 peptide

Pam3Cys.WNV001 be by Bachem bioscience limited company (BachemBioscience, Inc.) (King of Prussia, PA) synthetic.WNV001 is 20 amino acid peptides (SEQ ID NO:771) of Xi Niluo envelope protein, via the amino terminal serine residue of this peptide, chemically is coupled to three palmityl cysteine (Pam3Cys) parts.The chemistry of Pam3Cys.WNV001 is called [palmityl-Cys ((RS)-2,3-two (palmityl oxygen base)-propyl group)-LTSGHLKCRVKMEKLQLKGT (SEQ ID NO:771) acetate].The molecular mass of Pam3Cys.WNV001 is 3163.3 dalton.This peptide is to use solid phase synthesis process and FMOC chemosynthesis to get by Bachem.Synthetic by solid-phase peptide, the aminoacid sequence of Pam3Cys.WNV001 is combined on the chlorination H-Pro-2-chlorine trifluoromethyl resin.Peptide chain is increased by continuous coupling amino acid derivant.Before each coupling step is that a Fmoc-goes to protect step, and finishes by the repeated washing resin.Behind last amino acid derivativges of coupling, carry out last Fmoc-and go to protect step.At last, peptide resin is cleaned and drying under decompression.In solid-phase peptide between synthesis stage, carry out the color indicator test of each step, whether complete with monitoring Fmoc-cracking and follow-up amino acid derivativges coupling.Be peptide coupling, satisfy peptide moiety under I-hydroxybenzotriazole (HOBt) exists that with N, N '-dichloro hexyl-carbonyl imidodicarbonic diamide (DCCI) activates in advance with Pam3Cys-OH and growth.The solution that is become is filtered and adds to this peptide resin.When the response time finishes, peptide resin is cleaned and drying under decompression.Carry out the color indicator test, with the coupling of management and control Pam3Cys-OH.With the peptide finished by cultivating with trifluoroacetic acid (TFA) and solving from pitch shake.The product that will disengage (rough peptide material) is settled out and lyophilization from reactant mixture.Raw product is used for initial immunogenicity research.

Synthesizing of WNV-E peptide array

Peptide array (SEQ ID NOS:703-754) be by sigma Genosys (Wood blue this, TX) synthetic.

The result

The Xi Niluo fused protein

West nile virus (WNV) occurred in the area, temperate zone of Europe with North America in recent years, and the anxiety of popular and animal dead might take place in expression.The most serious phenomenon that WNV infects is a lethal encephalitis (brain inflammation) human and horse, and some poultry and wild birds death.In the U.S., WNV also has been the obvious origin cause of formation of human diseases.The envelope glycoprotein of Xi Niluo (WNV-E) may produce neutrality and protection antibody with other banzi virus.By with this antigen binding to clock sample receptors ligand, but compositions as herein described, fusion rotein and polypeptide target surely suitably antigen represent cell, and do not need adjuvant or other immunomodulator concoction.

As described herein, several strategies have been expected can assist in manufacturing west nile virus peplos (WNV-E) fusion rotein in the escherichia coli.The one motion is by with the proteic functional domain III of WNV-E (EIII), and the aminoacid of (optionally) functional domain II, merge to total length STF2, and transform a kind of less WNV-E antigen (for example, STF2.E, STF2.EIII+).Functional domain III is responsible for virus-host and interacts, and possesses many west nile virus neutralizing antibody epitopes.It also only contains and has in the total length envelope protein wherein 2 of 12 cysteine, makes it be easier to expression in escherichia coli.Second motion has been used to lack the hypermutation hinge region (for example STF2 Δ) of flagellin, produces less fused protein (STF2 Δ .EIII+) thus.It is needed that the hypervariable region of flagellin is not the TLR5 citation, and it removes the immunological competence that causes that easily may lower flagellin.STF2.EIII+ and STF2 Δ .EIII+ the two, get in expression in escherichia coli and purification all.Purified protein in bioanalysis, at its TLR5 citation living featuresization, and uses in the elisa assay of one group of many strain of WNV-E and neutralizing monoclonal antibody, shows characterization at its E epitope.The result of these researchs points out that STF2 Δ .EIII+ has higher PAMP activity, and has more the neutrality WNV-E epitope of configuration-sensitivity.

The purity of STF2.EIII+ and STF2 Δ .EIII+

Manufacturing and purification total are criticized STF2.EIII+ and STF2 Δ .EIII+ in escherichia coli.STF2.EIII+ expresses and is soluble protein, and uses the 4-step process to carry out purification under the non-denaturing condition, and it comprises anion-exchange chromatography art and gel filtration.From the ultimate output scope of 6L culture gained between 0.9mg to about 3.8mg, and all preparations with the endotoxin content that standard LAL program records low (pact＜0.1EU/mg protein, as described above).Otherwise STF2 Δ .EIII+ forms inclusion body in escherichia coli, and is to carry out purification under the degeneration condition.The tomography step of the purification STF2 Δ .EIII+ that is useful on all needs to use 8M carbamide.Behind purification, denatured protein is to be undertaken folding by the progressively dialysis that enables to remove gradually carbamide again.Folding again is to be to carry out under about 0.3mg/ml in protein concentration typically, can be owing to protein precipitation has loss.From two kinds of STF2 Δ .EIII+ preparations of 6L culture, produce about 1.2 and the protein of about 6.7mg, the two all has acceptable endotoxin degree.Person as expected, purified STF2.EIII+ and STF2 Δ .EIII+ move respectively on SDS PAGE and present about 65kDa and about 43kDa protein under reducing condition.Obviously, under reducing condition STF2 Δ .EIII+ move very fast a little.The mobility of this change may be because, the disulfide bond that relates to two cysteine residues among the envelope protein functional domain III forms.Again, detect bigger STF2 Δ .EIII+ by western blot analysis, its molecular weight is consistent with this proteic trimeric form (" the STF2 Δ .EIII+ of (STF2 Δ .EIII+) x3 or 3 units ").

The endotoxin degree and the TLR-5 activity of table 11:STF2.EIII+ (SEQ ID NO:658) and STF2 Δ .EIII+ (SEQ ID NO:674) fusion rotein

Mission Number

Protein

Output (mg)

Endotoxin degree (EU/ μ g)

TLR-5EC 50

052	STF2.EIII+	3.8	0.03	>5000.00ng/ml
					054	STF2.EIII+	0.9	0.02	1195.00ng/ml
057	STF2.EIII+	1.6	0.07	197.92ng/ml
					044	STF2Δ.EIII+	1.2	0.07	1.13ng/ml
045	STF2Δ.EIII+	6.7	0.07	4.34ng/ml

TLR5 activity in HEK293IL-8 analyzes

Be the PAMP activity of this two fusion rotein relatively, carry out the TLR5 bioanalysis then.With the HEK293IL-8 cell with two independently the serial dilutions of protein batch handle (Figure 62 A and 62B).Culture was cultivated 24 hours, collected conditioned medium and make with elisa assay IL-8.Shown in Figure 62 A, STF2 Δ .EIII+ represents effective TLR-5 activity.The regression analysis of titration curve is measured, the EC of crowd part 2004-044 and 2004-045 ₅₀Be respectively 1.13ng/ml and 4.34ng/ml (table 11, as described above).In this two case, the TLR5-specific activity exceeds 10-times at least than matched group Protein S TF2.OVA.On the contrary, 2 preparations of STF2.EIII+ present the remarkable more weak TLR5 activity than STF2.OVA.STF2.EIII+ criticizes the EC of part 054 and 057 ₅₀Be about 1195.00ng/ml and about 197.92ng/ml.

The antigenicity of STF2.EIII+ and STF2 Δ .EIII+

Be specific to the flagellin monoclonal antibody (6H11 of the N-end regions of STF2 by use, the Inotek pharmaceuticals, Bei Fuli, MA), and one group before be shown in vitro in and WNV-E-specific antibody (5C5,7H2,5H10,3A3 and 3D9, Bioreliance, the Lip river Ke Gu of west nile virus, MD) the direct ELISA that carries out, the antigenicity of check STF2.EIII+ and STF2 Δ .EIII+.Shown in Figure 63, relatively the reactivity of total length west nile virus envelope protein and STF2 Δ .EIII+ shows that west nile virus monoclonal antibody 5C5,5H10,3A3 and 7H2 (but non-3D9) discern this fused protein.5C5 in this reactive form and the EIII, 5H10,3A3 and 7H2 epitope infer the site unanimity.The epitope of 3D9 is the outside of dropping on west nile virus envelope protein functional domain III.Person as expected, all west nile virus monoclonal antibodies all with the reaction of total length west nile virus envelope protein, and the flagellin monoclonal antibody is only reacted with STF2 Δ .EIII+.This two protein all with many strains west nile virus peplos antiserum reaction, but STF2 Δ .EIII+ reactivity has slightly and lowers, and perhaps is owing to exist due to possible the epitope quantity minimizing in the minor feature territory.

Use 5C5 and 7H10 WNV monoclonal antibody, finish the antigenicity (Figure 64 A, 64B, 64C and 64D) of direct relatively STF2.EIII+ and STF2 Δ .EIII+.In these researchs, be with flat board coated with specified protein, then with many strains rabbit anti--E or mouse monoclonal antibody as described detect.Shown in Figure 64 A, 64B, 64C and 64D, STF2.EIII+ and STF2 Δ .EIII+ are all easily detected by the flagellin monoclonal antibody, and its reactivity there is no significant difference.Yet, observe reactive different with anti--peplos monoclonal antibody.The reactivity of STF2 Δ .EIII+ and 5C5 or 7H2 is significantly big with the STF2.EIII+ person of observing.Collective, these results represent that the flagellin 6H11 epitope of STF2 Δ .EIII+ is compromised, and suitable with the flagellin sequence of STF2.EIII+.They also emphasize these proteic functional domain III, and there were significant differences in the antigenicity aspect, and expression STF2 Δ .EIII+ contains more more important configuration dependency neutrality epitope than STF2.EIII+.

Effect and immunogenicity

Finished several and be used in the C3H/HeN mice, at the efficacy study of the protectiveness effect that is exciting check candidate in back with west nile virus through design.Research be typically by 5 groups in the 0th, 14 and 28 day, cause the mice (every group of 10 mices) of exempting from through intraperitoneal (i.p.) or subcutaneous (s.c.) and form.In the 21st and 35 day, the serum and test west nile virus envelope protein-IgG antibody (ELISA) of gathering, and in vitro and the ability (PRNT analysis) of west nile virus.In the 35th day, the WNV Strain 2741 of mice with fatal dose excited.In exciting back its survival of monitoring to reach 21 days.

Mice caused with STF2 Δ .EIII+i.p., 25 μ g STF2 Δ .EIII+s.c., 25 μ g STF2.EIII+i.p. and the 25 μ gSTF2.EIII+s.c of PBS, the fruit bat conditioned medium (CM, positive controls) that contains STF2.E, 25 μ g exempt from.West nile virus envelope protein antibody response, and survival data is through being shown in Figure 65 and 66.Before the 35th day, all groups of accepting STF2 Δ .EIII+ all have the west nile virus envelope protein IgG of significance degree.Otherwise the mice of accepting STF2.EIII+ does not have detectable west nile virus envelope protein antibody response.STF2 Δ .EIII+i.p. or s.c are given in throwing, can make to reach 100% survival after west nile virus excites.Conform to the immunogenicity of the difference of STF2.EIII+, when compared to the PBS matched group, candidate can provide few to the protectiveness of not having thus.In this research, immunogenicity and the effect of STF2.EIII+ are poor, are because due to the active attenuating of TLR5 and/or this proteinic weak EIII epitope reactivity.

Plaque reduces neutralization and tires

For further assessment by the caused west nile virus envelope protein of STF2 Δ .EIII+ antibody response, and with neutralization tire may be relevant the protection effect, carry out plaque then and reduce neutralization and test (PRNT).To taking from the 35th day serum sample of aforementioned efficacy study, test its ability that blocking-up west nile virus infects in the Vero cell of cultivating.Letter speech, will compile the mice serum sample deactivate through heat, and the twice serial dilution is in the PBS that contains 5% gelatin.To cultivate with the west nile virus strain 2741 of about 100pfu from the diluent that 1:10 begins.Virus/serum is mixed in about 37 ℃ cultivates 1h down, cultivate being paved with on Vero cell (ATCC, catalog number CCL-81, Manassas, the Virginia) monolayer in growing in two repetitions, six-hole tissue culturing plate then.Before adding 1% agar cover layer, shilling virus is adsorbed to this cell monolayer.Cell culture through infecting was cultivated 4 days down in 37 ℃, covered second cover layer that contains 4% dimethyl diaminophenazine chloride subsequently.The viral plaque of counting after 12 hours.Serum titer (the PRNT that record can cause 80% viral plaque number to reduce ₈₀).

In following table 12, list, derive from PRNT about the efficacy study of STF2.EIII+ and STF2 Δ .EIII+ ₈₀Data.In two independent studies that relate to STF2.EIII+, the survival rate of being reported is about 50% or still less, compile serum can't suppress plaque and form.Because the caused antibody response of this construct is faint, so find not unexpected.In three efficacy studies that relate to STF2 Δ .EIII+, survival rate is about 70% or higher, compile serum have neutralization and tire and be 1:40 or better.Neutralization is tired and is 1:40 or better, and protective effect is relevant typically with in vivo.

The survival and the PRNT of table 12:STF2.EIII+ (SEQ ID NO:658), STF2 Δ .EIII+ (SEQ ID NO:674) and STF2.E (SEQ ID NO:762) CM (matched group culture medium) fusion rotein ₈₀The result

Criticize part	Candidate	Approach	Research #	Survival (%)	PRNT 80(dilution factor)
						054	STF2.EIII+	i.p.	3	50	Negative
057	STF2.EIII+	i.p.	4	11	Negative
						057	STF2.EIII+	s.c.	4	20	Negative
044	STF2Δ.EIII+	i.p.	2	70	1:40
						045	STF2Δ.EIII+	i.p.	3	90	1:40
045	STF2Δ.EIII+	s.c.	3	100	1:160
						045	STF2Δ.EIII+	i.p.	4	100	1:80
045	STF2Δ.EIII+	s.c.	4	100	1:40
						--	STF2.ECM	i.p.	3	90	1:640
--	STF2.ECM	i.p.	4	--	1:1280

STF2 Δ .EIIIs+ is the modified version of STF2 Δ .EIII+

To be used for the STF2 Δ .EIII+ protein formulation that aforementioned mice efficacy study is tested, and, use progressively dialysis folding more subsequently by anion exchange and the size-eliminating tomography step purification that under the degeneration condition, carries out.With this processing procedure, produce two kinds and be equivalent to the monomer of STF2 Δ .EIII+ and the main species of trimeric form, and be mixture and exist in the end-product.For the inhomogeneities that makes end-product minimizes, developed preference then and made monomer or the folding again purification process that reaches of trimerical novelty.Do not form for active because know which kind of form of STF2 Δ .EIII+, or the two is all effectively not same, thus two species of milligram quantities produced, and test its effect in mice.

Originally and unclear why STF2 Δ .EIII+ is folding again can cause trimer to form.Yet when inspecting the sequence of STF2 Δ .EIII+ expression construct once more, we identify a cysteine residues in the peptide connexon sequence of separating STF2 Δ and EIII+.As if the existence of this cysteine can be disturbed, and the suitable disulfide bond during folding again forms, and may facilitate the trimeric form of STF2 Δ .EIII+.The cysteine that this is unnecessary changes over serine to use fixed point to cause change, and manufacturing and purification get modified protein (STF2 Δ .EIIIs+).It should be noted that will be somebody's turn to do the construct that replaces through serine folds again, only produces monomeric protein.

After exciting, in the C3H/HeN mice, assess the protectiveness effect of STF2 Δ .EIII+ (monomer) and STF2 Δ .EIIIs+ (trimer) with west nile virus.Five groups of mices (every group of 10 mices) in the 0th, 14 and 28 day, are caused with about 25ug protein through s.c. and to exempt from.In the 21st and 35 day, the serum and test WNV-E IgG antibody (ELISA) of gathering.In the 38th day, the WNV Strain 2741 of mice with fatal dose excited, and monitor its survival and reach 21 days.(the 35th day, ELISA result Figure 67) and survival data (Figure 68) pointed out that all constructs all cause the reactive IgG of the WNV-E of significance degree before virus excites, and provide about 90% to the deadly protective effect of infecting of about 100% antagonism by short liter 2.These find indication, and the monomer of STF2 Δ .EIII+ or polymer (for example trimer) form is effectively, and extra cysteine is removed from this construct, can't have perception ground to impact its effect.Remove the cysteine in the peptide connexon sequence, can be by lowering the inhomogeneities behind protein refolding, and simplify protein purification.

Conclusion

Produced two kinds and contained salmonella typhimurium flagellin (STF2) through merging to the recombinant fusion protein of the EIII+ functional domain of west nile virus envelope protein.One comprises total length STF2 sequence (STF2.EIII+), and another person comprises that it lacks the modified version of STF2 (STF2 Δ .EIII+) of the inner hypervariable region of STF2.This two protein is in expression in escherichia coli, and gets by the prior art method purification that uses anion-exchange chromatography art and gel filtration.STF2.EIII+ is soluble protein to make, and carries out purification under the non-denaturing condition.Otherwise STF2 Δ .EIII+ expresses and is soluble protein, and carries out purification under the degeneration condition, and is undertaken folding to remove carbamide by progressively dialysing again.Analyze in HEK293IL8, the preparation of STF2 Δ .EIII+ shows the TLR-5 activity big than STF2.EIII+.

In the envelope protein antigen epitope display that uses elisa assay and west nile virus envelope protein antibody was analyzed, STF2 Δ .EIII+ showed more more more important configuration dependency neutrality epitope.Meet by observed effective TLR-5 activity of STF2 Δ .EIII+ and envelope protein antigen epitope antigen, STF2 Δ .EIII+ has hyperimmunization originality and effectiveness in the mice that the west nile virus through fatal dose excites.Because the monomer of STF2 Δ .EIII+ and trimer species are to produce, the cysteine in the peptide connexon sequence of expression construct is changed over serine in this protein purification program.Remove this cysteine and can eliminate this proteic trimeric form of generation during folding, and cause resulting from the monomeric products that in vivo represents effective effect.

Japanese encephalitis's fusion rotein

JE virus is distributed in Asia and northern Australia (wherein about 10,000 death take place annual about 50,000 cases).Nearest approved inactivated virus vaccine is relevant with acute case of disseminating encephalomyelitis, and excitation Japan is healthy, this vaccine of national popularization is advised by labourer and social work department.Because at infected mice brain, or even in cell culture, make the program complexity of inactivation of viruses, and the harmful incident relevant with inactivation of viruses may take place, so cause the chance based on the JE vaccine of recombinant.

Make up STF2 Δ .JEIII+ fusion constructs.Produce JE EIII+DNA fragment with synthesis mode, and be used for escherichia coli expression through the codon optimization.This sequence is engaged in the pET24STF2 Δ, and produces pETSTF2 Δ .JEIII+.Expression construct is carried out screening by restriction analysis, and for inducing in the expression of escherichia coli BLR (DE3) by IPTG.Confirm the DNA sequence of each construct, and enlarged this proteinic manufacturing scale.Produced a collection of material.Purification gets about altogether 24mg material.This material has effective TLR activity, and endotoxin of acceptable degree (about 0.03EU/ μ g) and A280/A260 ratio are about 1.3.

The jaundice phallotoxins

The evaluation of WNV-E specific antibody epitope

For identifying in the west nile virus envelope protein, can produce several peptide arrays then by deriving from the linear epitope that STF Δ .EIIIs+ causes the antiserum identification of exempting from mice.Wherein an array is by the whole west nile virus functional domain III of leap, and the length of partial function territory II is formed (SEQID NOS:728-754) by 20 amino acid whose overlapping peptides.The ELISA that carries out with this array identify a kind of 20 aminoacid sequences with high response, it is defined as the N-end regions of functional domain III and comprises partial function territory I functional domain CRVKMEKLQLKGTTYGVCSK (SEQ ID NO:728) through figure shop.For with this epitope close mapping, satisfy the extra array (SEQ ID NOS:703-754) that generation concentrates on functional domain I and II joint.These arrays comprise that alanine replaces scanning, are important aminoacid (SEQ IDNOS:728-754) to identify for antibodies.Shown in Figure 69 and 70, must hang oneself STF2 Δ .EIII (monomer and trimer) and STF2 Δ .EIIIs+ causes the antiserum of exempting from mice, can engage (peptide E-30 to E-42) with crossing over EI/EIII, and comprise the reactive polypeptide of E2-21 peptide CRVKMEKLQLKGTTYGVCSK (SEQ ID NO:728).When specific amino acids (E6, K7, L10 and K11) is changed into alanine, this reactive serious lower (Figure 71).Though do not know to discern whether the antibody of this epitope is neutrality, still be devoted to design and test this regional peptide vaccine based on WNV-E.

The immunity of Pam3Cys.WNV001 peptide vaccine

Use 20 aminoacid sequence LTSGHLKCRVKMEKLQLKGT (SEQ ID NO:772) (Pu Nake, people such as R., vaccine 23:4442-4452 (2005)) synthetic, merge fat west nile virus envelope protein to Pam3Cys in the N-end.The immunogenicity of testing this peptide in the C3H/HeN mice, and with the peptide that does not contain Pam3Cys relatively (Figure 72).By as the described direct ELISA of example, test to such an extent that hang oneself and cause the sero-fast reactivity of exempting from animal, and the result point out that the Pam3Cys.WNV001 peptide significantly has more immunogenicity without the peptide that TLR2 modifies.Derive from the antiserum of these researchs, will in viral neutralization analysis (PRNT), test, whether can be in vitro and west nile virus to measure the antibody caused.Also will excite in the model through the fat peptide and to test, to analyze its antagonism protection effect that virus excites that causes death in west nile virus.

Analyze development

Competitive ELISA analysis development

For analyzing derived from exempting from mice sero-fast and potential through causing, then use it can be in culture and west nile virus, and with the configuration-sensitivity epitope reaction in the functional domain III of west nile virus envelope protein antigen, very characterization monoclonal antibody (7H2) develop the competitive ELISA analysis.This analyzes through being designed to, and a kind ofly measures to such an extent that hang oneself and cause the ELISA that catches that the serum of exempting from animal prevents 7H2 and the bonded ability of west nile virus envelope protein.To take from efficacy study 4 (Figure 65 and Figure 66, the scope of the 35th day mouse resisting anteserum table 13) is from the serial dilutions of 1:10 to 1:5000, cultivate with biotinylation west nile virus envelope protein, add to then in advance coated with 7H2 monoclonal antibody (Bioreliance, Lip river Ke Gu, elisa plate MD).After cleaning the unconjugated material of removal for several times, use affinity element-HRP to detect bonded west nile virus envelope protein.Result by representativeness experiment gained is shown in Figure 69.Under the 1:25 dilution factor, when test derived from when STF2 Δ .EIIIs causes the antiserum of exempting from animal, observe combining of west nile virus envelope protein and 7H2-coated board, measurable loss is arranged.To replace antigenic imitation and cause the antiserum of exempting from mice, then there is not the competition of detecting derived from accepting PBS.These PRELIMINARY RESULTS prove, the antibody that is caused by STF2 Δ .EIII+ and 7H2 compete and the combining of west nile virus envelope protein.These find to exempt from observed arriving in the animal with being obtained to cause through STF2 Δ .EIII+, can avoid the protective effect that WNV infects and conform to, and help to set up in vitro the reactive and association between effect in vivo of antibody antigen epi-position.

Though the present invention lists especially and describes with reference to example embodiment, practise in this skill personage should be appreciated that, depart under the scope that appended claims of the present invention contain, in nothing in wherein carrying out various changes in form and details.

Claims (157)

1. make the method for protein that stimulates individual interior protective immunological reaction for one kind, it comprises the following step:

A) a proteinic part is isolated to form protein portion thus from the natural viral hemagglutinin, wherein this protein portion comprises

I) at least a portion of ball head, and

Ii) at least a at least a portion that makes ball head in fact still possess the secondary structure of its tertiary structure,

And wherein this protein portion lacks film fusion function territory, strides film functional domain and kytoplasm functional domain;

B) will the encode nucleotide sequence of this protein portion is converted in the prokaryotic host cell; And

C) cultivate this prokaryotic host cell and make the protein that stimulates individual interior protective immunological reaction thus.

2. method according to claim 1, wherein, described protein portion further lacks signal sequence.

3. method according to claim 1, wherein, described secondary structure comprises at least a portion of at least one beta sheet.

4. method according to claim 1, wherein, described secondary structure comprises at least one α spiral.

5. method according to claim 1, wherein, described secondary structure protein portion further comprises at least a member who is selected from the cohort of being made up of salt bridge, leucine zipper and zinc fingers.

6. method according to claim 1, wherein, described protein portion further comprises at least one disulfide bond.

7. method according to claim 1, wherein, described protein portion further comprises at least two disulfide bond.

8. method according to claim 1, wherein, described protein portion further comprises at least four disulfide bond.

9. method according to claim 1, wherein, described protein portion further comprises at least five disulfide bond.

10. method according to claim 1, wherein, described protein portion further comprises at least six disulfide bond.

11. method according to claim 1, it comprises that further at least one is selected from the nucleotide sequence of the amino acid residue of the cohort of being made up of hydrophilic amino-acid residue, polar amino acid residue and neutral amino acid residue to encode, and replaces the step of the nucleotide sequence of at least one hydrophobic amino acid residues in this protein portion of coding.

12. method according to claim 11, wherein, described hydrophobic amino acid residues comprises at least a member who is selected from the cohort of being made up of phenylalanine residue, trp residue and tyrosine residue.

13. method according to claim 11, wherein, described polar amino acid residue comprises at least a member who is selected from the cohort of being made up of asparagicacid residue and glutaminic acid residue.

14. method according to claim 1, wherein, described viral hemagglutination element comprises influenza virus haemagglutinin protein.

15. method according to claim 14, wherein, described influenza virus haemagglutinin protein comprises influenza A virus hemagglutinin protein.

16. method according to claim 15, wherein, described influenza A virus is at least a member who is selected from the cohort of being made up of H1, H2, H3, H5, H7 and H9.

17. method according to claim 14, wherein, described influenza virus haemagglutinin protein comprises influenza B viral hemagglutination fibroin matter.

18. method according to claim 14, wherein, described influenza virus haemagglutinin protein comprises influenza C viral hemagglutination fibroin matter.

19. method according to claim 1, wherein, described prokaryotic host cell comprises the escherichia coli prokaryotic host cell.

20. method according to claim 1, wherein, described prokaryotic host cell comprises the Rhodopseudomonas prokaryotic host cell.

21. method according to claim 1, wherein, described prokaryotic host cell comprises the bacillus prokaryotic host cell.

22. method according to claim 1, wherein, described prokaryotic host cell comprises the salmonella spp prokaryotic host cell.

23. method according to claim 1, it further comprises the step that transforms prokaryotic host cell with the molecular chaperones nucleotide sequence.

24. method according to claim 23, wherein, described molecular chaperones nucleotide sequence is at least a member who is selected from the cohort of being made up of groES-groEL molecular chaperones, dnaK-dnaJ-grpE molecular chaperones, groES-groEL-tig molecular chaperones and tig molecular chaperones.

25. method according to claim 1, it comprises that further at least a portion with clock sample receptor stimulating agent merges to this proteinic step.

26. method according to claim 25, wherein, described clock sample receptor stimulating agent comprises at least a member who is selected from the cohort of being made up of clock sample receptor 2 agonists, clock sample receptor 4 agonist and clock sample receptor 5 agonist.

27. method according to claim 1, it comprises that further nucleotide sequence operability with at least a portion of coding clock sample receptor stimulating agent is linked to the step of nucleotide sequence of the protein portion of coding viral hemagglutination element.

28. method according to claim 27, it further comprises connexon is inserted in step between this clock sample receptor stimulating agent and this protein portion.

29. method according to claim 27, wherein, described clock sample receptor stimulating agent comprises clock sample receptor 5 agonist.

30. method according to claim 29, wherein, described clock sample receptor 5 agonist comprise at least a portion of flagellin.

31. method according to claim 30, wherein, described flagellin lacks at least a portion of hinge region.

32. method according to claim 30, wherein, described flagellin comprises at least a member who is selected from the cohort of being made up of salmonella typhimurium flagellin, coli flagellum albumen, Munich Salmonella flagellin, yersinia flagellin, Campylobacter flagellin, bacillus pyocyaneus flagellin and Listeria monoeytogenes flagellin.

33. method according to claim 1, it further comprises the step that the proteic nucleotide sequence operability of code carrier is linked to the nucleotide sequence of this protein portion of coding.

34. method according to claim 33, wherein, described carrier protein is at least a member who is selected from by B subunit, coat protein for mosaic virus of tobacco, rabies virus envelope protein, rabies virus envelope glycoprotein, Elityran, heat shock protein 60, keyhole-limpet hemocyanin and early stage secretion antigen tuberculosis-6 cohorts of being formed of the cross reactivity saltant of tetanus toxoid, vibrio cholera toxoid, diphtheria toxoid, diphtheria toxoid, escherichia coli thermal instability enterogenous endotoxin

35. method according to claim 1, it further comprises at least a portion of carrier protein is merged step to this protein portion.

36. make the method for protein that stimulates individual interior protective immunological reaction for one kind, it comprises the following step:

A) a proteinic part is isolated from the natural viral hemagglutinin formed protein portion thus, wherein, described protein portion comprises

I) at least a portion of ball head, and

Ii) at least a at least a portion that makes ball head in fact still possess the secondary structure of its tertiary structure,

And wherein, described protein portion lacks film fusion function territory, strides film functional domain and kytoplasm functional domain;

B) will the encode nucleotide sequence of this protein portion changes and to infect to the eucaryon host cell, and wherein, described eukaryotic host cell is not the pichia pastoris phaff eukaryotic host cell; And

C) cultivate this eukaryotic host cell and make the protein that stimulates individual interior protective immunological reaction thus.

37. method according to claim 36, wherein, described protein portion further lacks signal sequence.

38. method according to claim 36, wherein, described protein portion comprises the sialic acid binding site.

39. method according to claim 36, wherein, described secondary structure comprises at least a portion of at least one beta sheet.

40. method according to claim 36, wherein, described secondary structure comprises at least a member who is selected from the cohort of being made up of salt bridge and α spiral.

41. method according to claim 36, it comprises that further at least one is selected from the nucleotide sequence of the amino acid residue of the cohort of being made up of hydrophilic amino-acid residue, polar amino acid residue and neutral amino acid residue to encode, and replaces the nucleotide sequence of at least one hydrophobic amino acid residues in this protein portion of coding.

42. method according to claim 36, it further comprises the step of at least one saccharifying site disappearance of the nucleotide sequence that will be arranged in this protein portion of encoding.

43. according to the described method of claim 42, wherein, described saccharifying site comprises N-saccharifying site.

44. method according to claim 36, wherein, described eukaryotic host cell comprises the Saccharomyces eukaryotic host cell.

45. method according to claim 36, wherein, described eukaryotic host cell comprises the fungus eukaryotic host cell.

46. method according to claim 36, wherein, described eukaryotic host cell comprises the parasite eukaryotic host cell.

47. according to the described method of claim 46, wherein, described eukaryotic host cell comprises the leishmania eukaryotic host cell.

48. according to the method for claim 36, wherein, described eukaryotic host cell comprises the Kluyveromyces lactis host cell.

49. method according to claim 36, it further comprises at least a portion of clock sample receptor stimulating agent is merged step to this protein portion.

50. according to the described method of claim 49, wherein, described clock sample receptor stimulating agent comprises at least a member who is selected from the cohort of being made up of clock sample receptor 2 agonists, clock sample receptor 4 agonist and clock sample receptor 5 agonist.

51. method according to claim 36, it further comprises the step that the nucleotide sequence operability of at least a portion of coding clock sample receptor stimulating agent is linked to the nucleotide sequence of this protein portion of coding.

52. according to the described method of claim 51, it further comprises connexon is inserted in step between this clock sample receptor stimulating agent and this protein portion.

53. according to the described method of claim 51, wherein, described clock sample receptor stimulating agent comprises clock sample receptor 5 agonist.

54. according to the described method of claim 53, wherein, described clock sample receptor 5 agonist comprise at least a portion of flagellin.

55. according to the described method of claim 54, wherein, described flagellin lacks at least a portion of hinge region.

56. according to the described method of claim 54, it further comprises the step of at least one saccharifying site disappearance of the nucleotide sequence that will be arranged in the flagellin of encoding.

57. according to the described method of claim 54, wherein, described flagellin comprises at least a member who is selected from the cohort of being made up of salmonella typhimurium flagellin, coli flagellum albumen, Munich Salmonella flagellin, yersinia flagellin, Campylobacter flagellin, bacillus pyocyaneus flagellin and Listeria monoeytogenes flagellin.

58. method that stimulates individual interior protective immunity; it comprises and comprises that with a kind of the compositions administration of the protein portion that derives from the natural viral hemagglutinin gives this individual step; wherein; described protein portion comprises at least a portion of ball head; and a kind of at least a portion that makes ball head in fact still possess the secondary structure of its tertiary structure; and wherein, described protein portion lacks film fusion function territory, strides film functional domain and kytoplasm functional domain.

59. a method of making the viral hemagglutination fibroin that stimulates individual interior protective immunological reaction, it comprises the following step:

A) a proteinic part is isolated from the natural viral hemagglutinin formed protein portion thus, wherein, described protein portion comprises

I) at least a portion of ball head, and

Ii) at least a at least a portion that makes ball head in fact still possess the secondary structure of its tertiary structure,

And wherein, described protein portion lacks film fusion function territory, strides film functional domain and kytoplasm functional domain;

B) will the encode nucleotide sequence of this protein portion changes and to infect to the eucaryon host cell, and wherein, described eukaryotic host cell is not the insect host cell of pichia pastoris phaff eukaryotic host cell or stable transfection; And

C) cultivate this eukaryotic host cell and make thus this stimulation individual in the protein of protective immunological reaction.

60. a method of making the viral hemagglutination fibroin that stimulates individual interior protective immunological reaction, it comprises the following step:

A) nucleotide sequence that prokaryotic host cell is striden the viral hemagglutination element of film functional domain and kytoplasm functional domain with at least a shortage of encoding transforms; And

B) cultivate this prokaryotic host cell and make thus this stimulation individual in the protein of protective immunological reaction.

61. according to the described method of claim 60, wherein, described viral hemagglutination element comprises influenza virus haemagglutinin protein.

62. according to the described method of claim 60, it further comprises the step that prokaryotic host cell is transformed with the molecular chaperones nucleotide sequence.

63. according to the described method of claim 60, it further comprises the step that the nucleotide sequence operability of at least a portion of coding clock sample receptor stimulating agent is linked to the nucleotide sequence of this protein portion of coding.

64. method that stimulates individual interior protection immunity; it comprises and comprises that with a kind of having a kind of shortage strides the proteinic compositions administration of the viral hemagglutination element of film functional domain and kytoplasm functional domain and give this individual step; wherein, described protein expression is in prokaryotic cell.

65. according to the described method of claim 64, it gives in this individual compositions in administration and further comprises carrier protein.

66. compositions, it comprises at least a portion of the molecule pattern of at least a pathogen-connection, and the protein portion of natural viral hemagglutinin, wherein, the protein portion of described natural viral hemagglutinin comprises at least a portion and at least a at least a portion that makes ball head in fact still possess the secondary structure of its tertiary structure of ball head, and wherein, the part of described natural viral hemagglutinin lacks film fusion function territory, strides film functional domain and kytoplasm functional domain.

67. according to the described compositions of claim 66, it further comprises carrier protein.

68. according to the compositions of claim 66, wherein, the molecule pattern of described pathogen-connection and the part of natural viral hemagglutinin are the composition of fused protein.

69. according to the described compositions of claim 66, wherein, the molecule pattern of described pathogen-connection comprises clock sample receptor stimulating agent.

70. according to the described compositions of claim 69, wherein, described clock sample receptor stimulating agent comprises at least a member who is selected from the cohort of being made up of clock sample receptor 2 agonists, clock sample receptor 4 agonist and clock sample receptor 5 agonist.

71. according to the described compositions of claim 70, wherein, described clock sample receptor stimulating agent comprises clock sample receptor 5 agonist.

72. according to the described compositions of claim 71, wherein, described clock sample receptor 5 agonist comprise flagellin.

73. according to the described compositions of claim 72, wherein, described flagellin comprises at least a member who is selected from the cohort of being made up of salmonella typhimurium flagellin, coli flagellum albumen, Munich Salmonella flagellin, yersinia flagellin, Campylobacter flagellin, bacillus pyocyaneus flagellin and Listeria monoeytogenes flagellin.

74. a compositions that comprises it for the flagellin composition of at least a portion of flagellin, wherein, described flagellin composition comprises at least one cysteine residues, and makes this flagellin composition activation clock sample receptor 5 thus.

75. according to the described compositions of claim 74, it further comprises at least a at least a portion that is selected from the member of the cohort of being made up of clock sample receptor 1 agonist, clock sample receptor 2 agonists, clock sample receptor 3 agonist, clock sample receptor 4 agonist, clock sample receptor 6 agonist, clock sample receptor 7 agonist, clock sample receptor 8 agonist and clock sample receptor 9 agonist.

76. according to the described compositions of claim 75, wherein, described clock sample receptor 2 agonists is Pam3Cys.

77. according to the described compositions of claim 76, wherein, described Pam3Cys further comprises the cysteine that at least one is extra.

78. according to the described compositions of claim 74, wherein, at least one cysteine residues replaces at least one amino acid residue in the natural flagellin aminoacid sequence that has this flagellin composition.

79. according to the described compositions of claim 78, wherein, described cysteine residues is at least one aminoacid away from clock sample receptor 5 recognition sites of this flagellin composition.

80. according to the described compositions of claim 74, wherein, described flagellin composition comprises at least a portion and the combination of this cysteine residues of natural flagellin aminoacid sequence.

81. 0 compositions according to Claim 8, wherein, described cysteine residues is at least a member who is selected from the cohort of being made up of the carboxyl-end amino acid of the amino-end amino acid of this flagellin composition and this flagellin composition.

82. 0 described compositions according to Claim 8, wherein, described cysteine residues is at least one aminoacid away from clock sample receptor 5 recognition sites of this flagellin composition.

83. according to the described compositions of claim 74, wherein, described flagellin composition lacks at least a portion of hypervariable region.

84. according to the described compositions of claim 74, it further comprises a kind of antigenic at least a portion.

85. 4 described compositionss according to Claim 8, wherein, described antigen chemically conjugation to the flagellin composition.

86. 5 described compositionss according to Claim 8, wherein, described antigen is essentially hydrophobicity.

87. 6 described compositionss according to Claim 8, wherein, described chemical conjugation causes this antigenic water solublity as the composition of compositions to increase.

88. according to the described compositions of claim 74, wherein, described cysteine residues is arranged in the hypervariable region of this flagellin composition.

89. according to the described compositions of claim 74, wherein, described flagellin is at least a at least a portion that is selected from the member of the cohort of being made up of salmonella typhimurium flagellin, escherichia coli fliC flagellin, Munich Salmonella flagellin, bacillus pyocyaneus flagellin and Listeria monoeytogenes flagellin.

90. 9 described compositionss according to Claim 8, wherein, described flagellin is at least a portion of salmonella typhimurium flagellin, and this salmonella typhimurium flagellin is to be selected from the cohort of being made up of SEQID NO:812 and SEQ ID NO:816.

91. according to the described compositions of claim 90, wherein at least one cysteine residues replaces at least one amino acid residue in the natural flagellin aminoacid sequence that has this flagellin composition.

92. according to the described compositions of claim 91, wherein, described aminoacid is selected from by the amino acid/11,237,238,239,240 of SEQ ID NO:812,241 and the aminoacid of 495 cohorts of being formed at least one.

93. according to the described compositions of claim 90, wherein, described flagellin is at least a portion of salmonella typhimurium flagellin, and this salmonella typhimurium flagellin comprises at least a portion of SEQ IDNO:816.

94. according to the described compositions of claim 93, wherein at least one cysteine residues replaces at least one amino acid residue in the natural flagellin aminoacid sequence that has this flagellin composition.

95. according to the described compositions of claim 94, wherein, described aminoacid is selected from by the amino acid/11,240,241,242,243 of SEQ ID NO:816,244 and the aminoacid of 505 cohorts of being formed for few one.

96. 9 described compositionss according to Claim 8, wherein, described flagellin is at least a portion of Munich Salmonella flagellin, and this Munich Salmonella flagellin comprises at least a portion of SEQ IDNO:504.

97. according to the described compositions of claim 96, wherein at least one cysteine residues replaces at least one amino acid residue in the natural flagellin aminoacid sequence that has this flagellin composition.

98. according to the described compositions of claim 97, wherein, described aminoacid is selected from by the amino acid/11,237,238,239,240 of SEQ ID NO:504,241 and the aminoacid of 504 cohorts of being formed for few one.

99. 9 described compositionss according to Claim 8, wherein, described flagellin is at least a portion of bacillus pyocyaneus flagellin, and this bacillus pyocyaneus flagellin comprises at least a portion of SEQ ID NO:815.

100. according to the described compositions of claim 99, wherein at least one cysteine residues replaces at least one amino acid residue in the natural flagellin aminoacid sequence that has this flagellin composition.

According to the described compositions of claim 100, wherein, described aminoacid is selected from by the amino acid/11,211,212 of SEQ ID NO:815,213 and the aminoacid of 393 cohorts of being formed for few one.

9 described compositionss according to Claim 8, wherein, described flagellin comprises at least a portion of Listeria monoeytogenes flagellin, and this Listeria monoeytogenes flagellin comprises at least a portion of SEQ ID NO:820.

According to the described compositions of claim 102, wherein at least one cysteine residues replaces at least one amino acid residue in the natural flagellin aminoacid sequence that has this flagellin composition.

According to the described compositions of claim 103, wherein, described aminoacid is selected from by the amino acid/11,151,152,153 of SEQ ID NO:820,154 and the aminoacid of 287 cohorts of being formed for few one.

9 described compositionss according to Claim 8, wherein, described flagellin is the proteic at least a portion of coli flagellum, and this coli flagellum albumen comprises at least a portion of SEQ ID NO:502.

According to the described compositions of claim 105, wherein at least one cysteine residues replaces at least one amino acid residue in the natural flagellin aminoacid sequence that has this flagellin composition.

According to the described compositions of claim 106, wherein, described aminoacid is selected from by the amino acid/11,238,239,240,241,242 of SEQ ID NO:502,243 and the aminoacid of 497 cohorts of being formed for few one.

4 described compositionss according to Claim 8, wherein, described antigen is at least a portion of influenza antigen.

According to the described compositions of claim 108, wherein, described influenza antigen is at least a portion of influenza virus conformability memebrane protein.

110. according to the described compositions of claim 108, wherein, described influenza antigen is an influenza virus A antigen.

111. according to the described compositions of claim 108, wherein, described influenza antigen is an influenza virus B antigen.

112. according to the described compositions of claim 108, wherein, described influenza antigen is an influenza virus C antigen.

113. according to the described compositions of claim 109, wherein, described conformability memebrane protein is the proteic at least a portion of hemagglutinin.

114. according to the described compositions of claim 113, wherein, the proteic part of described hemagglutinin comprises at least a portion of the proteic ripe shearing site of hemagglutinin.

115. according to the described compositions of claim 114, wherein, described ripe shearing site comprises less a kind of member who is selected from the cohort of being made up of SEQ ID NO:530, SEQ ID NO:532, SEQ ID NO:533, SEQ ID NO:534, SEQ ID NO:535, SEQ ID NO:600, SEQ ID NO:601, SEQ ID NO:602, SEQ ID NO:603, SEQ ID NO:821, SEQ ID NO:796, SEQ ID NO:797, SEQ IDNO:798, SEQ ID NO:799 and SEQ ID NO:822.

116. according to the described compositions of claim 109, wherein, described conformability memebrane protein comprises substrate 2 proteic at least a portion.

117. 4 described compositionss according to Claim 8, wherein, described antigen is at least a at least a portion that is selected from the member of the cohort of being made up of west nile virus albumen, blue good special virus protein, elder brother Tianjin virus protein, Murray Valley encephalitis virus protein, Japanese encephalitis virus albumen, tick-brone encephalitis virus albumen, dengue fever 1 virus protein, dengue fever 2 virus proteins, dengue fever 3 virus proteins, dengue fever 4 virus proteins, hepatitis C virus albumen and yellow fever virus albumen.

118. 4 described compositionss according to Claim 8, wherein, described antigen comprises the antigen that pathogen is relevant.

119. according to the described compositions of claim 118, wherein, the antigen that described pathogen is relevant is at least a member who is selected from the cohort of being made up of respiratory tract amalgamation virus, Plasmodium falciparum and Plasmodium vivax.

120. according to the described compositions of claim 74, it further comprises carrier protein.

121. according to the described compositions of claim 120, wherein, described carrier protein is at least a member who is selected from by B subunit, coat protein for mosaic virus of tobacco, rabies virus envelope protein, rabies virus envelope glycoprotein, Elityran, heat shock protein 60, keyhole-limpet hemocyanin and early stage secretion antigen tuberculosis-6 cohorts of being formed of the cross reactivity saltant of tetanus toxoid, vibrio cholera toxoid, diphtheria toxoid, diphtheria toxoid, escherichia coli thermal instability enterogenous endotoxin.

122. according to the described compositions of claim 74, the wherein at least a member who is selected from the cohort of being made up of arginine residues, serine residue and histidine residues replaces at least a at least one lysine that is selected from the member of the cohort of being made up of at least a portion of at least a portion of flagellin composition and flagellin.

123. compositions, it comprises that it is the clock sample receptor stimulating agent composition of at least a portion of clock sample receptor stimulating agent, wherein, described clock sample receptor stimulating agent is formed and is comprised at least one cysteine residues, be arranged in natural clock sample receptor stimulating agent and locating of cysteine residues do not occur, and make this clock sample receptor stimulating agent form activation clock sample receptor thus.

124. according to the described compositions of claim 123, wherein, described clock sample receptor stimulating agent is formed at least a portion and the combination of this cysteine residues that comprises natural clock sample receptor stimulating agent.

125. according to the described compositions of claim 124, wherein, described cysteine residues is at least one aminoacid away from the clock sample receptor recognition site of clock sample receptor stimulating agent.

126. according to the described compositions of claim 123, wherein, described clock sample receptor stimulating agent is at least a at least a portion that is selected from the member of the cohort of being made up of clock sample receptor 1 agonist, clock sample receptor 2 agonists, clock sample receptor 3 agonist, clock sample receptor 4 agonist, clock sample receptor 5 agonist, clock sample receptor 6 agonist, clock sample receptor 7 agonist, clock sample receptor 8 agonist and clock sample receptor 9 agonist.

127. according to the described compositions of claim 126, it further comprises at least a antigenic at least a portion.

128. a compositions, it comprises its flagellin composition at least a portion of flagellin, and wherein, at least one lysine of described flagellin composition is replaced by at least one arginine, makes flagellin composition activation clock sample receptor 5 thus.

129. according to the described compositions of claim 128, wherein, described flagellin is at least a at least a portion that is selected from the member of the cohort of being made up of salmonella typhimurium flagellin, escherichia coli fliC flagellin, Munich Salmonella flagellin, bacillus pyocyaneus flagellin and Listeria monoeytogenes flagellin.

130. according to the described compositions of claim 129, wherein, described flagellin is at least a portion of salmonella typhimurium flagellin, and this salmonella typhimurium flagellin is to be selected from the cohort of being made up of SEQ ID NO:812 and SEQ ID NO:816.

131. according to the described compositions of claim 130, wherein, the described lysine residue that replaces through arginine is selected from by the lysine 19,41,58,135,160,177,179,203,215,221,228,232,241,251,279,292,308,317,326,338,348,357,362,369,378,384,391 of SEQ ID NO:812 and the lysine of 410 cohorts of being formed at least one.

132. according to the described compositions of claim 130, wherein, the described lysine residue that replaces through arginine is selected from by the lysine 20,42,59,136,161,177,182,189,209,227,234,249,271,281,288,299,319,325,328,337,341,355,357,369,381,390,396,403,414 of SEQ ID NO:816 and the lysine of 422 cohorts of being formed at least one.

133. according to the described compositions of claim 129, wherein, described flagellin is an escherichia coli fliC flagellin, and this coli flagellum albumen comprises at least a portion of SEQ ID NO:814.

134. according to the described compositions of claim 129, wherein, described flagellin is at least a portion of Munich Salmonella flagellin, and this Munich Salmonella flagellin comprises at least a portion of SEQID NO:813.

135. according to the described compositions of claim 129, wherein, described flagellin is at least a portion of bacillus pyocyaneus flagellin, and this bacillus pyocyaneus flagellin comprises at least a portion of SEQ ID NO:815.

136. according to the described compositions of claim 129, wherein, described flagellin is at least a portion of Listeria monoeytogenes flagellin, and this Listeria monoeytogenes flagellin comprises at least a portion of SEQ ID NO:500.

137. according to the described compositions of claim 128, it further comprises at least a antigenic at least a portion.

138. according to the described compositions of claim 137, wherein, described antigen is after lysine replaces with arginine, conjugation is to the flagellin composition.

139. according to the described compositions of claim 137, wherein, described antigen is at least a portion of influenza antigens.

140. according to the described compositions of claim 139, wherein, described influenza antigens is at least a portion of conformability memebrane protein.

141. according to the described compositions of claim 140, wherein, described conformability memebrane protein comprises the proteic at least a portion of hemagglutinin.

142. according to the described compositions of claim 140, wherein, described conformability memebrane protein comprises substrate 2 proteic at least a portion.

143. according to the described compositions of claim 139, wherein, described influenza antigen is an influenza virus A antigen.

144. according to the described compositions of claim 139, wherein, described influenza antigen is an influenza virus B antigen.

145. according to the described compositions of claim 139, wherein, described influenza antigen is an influenza virus C antigen.

146. a compositions, it comprises its flagellin composition at least a portion of flagellin, and wherein, at least one lysine of described flagellin composition is replaced by at least one serine, makes flagellin composition activation clock sample receptor 5 thus.

147. a compositions, it comprises its flagellin composition at least a portion of flagellin, and wherein, at least one lysine of described flagellin composition is replaced by at least one histidine, makes flagellin composition activation clock sample receptor 5 thus.

148. one kind stimulates individual interior immunoreactive method, it comprises this individual step is given in a kind of compositions administration of flagellin composition that it is included as at least a portion of flagellin, wherein, described flagellin composition comprises at least one cysteine residues, and makes flagellin composition activation clock sample receptor 5 thus.

149. according to the described method of claim 148, it further comprises at least a antigenic at least a portion.

150. one kind stimulates individual interior immunoreactive method, it comprises this individual step is given in a kind of compositions administration of flagellin composition that it is included as at least a portion of flagellin, wherein, at least one lysine of described flagellin composition is replaced by at least one arginine, makes flagellin composition activation clock sample receptor 5 thus.

151. according to the described method of claim 150, it further comprises at least a antigenic at least a portion.

152. one kind stimulates individual interior immunoreactive method, it comprises this individual step is given in a kind of compositions administration of flagellin composition that it is included as at least a portion of flagellin, wherein, at least one lysine of described flagellin composition is replaced by at least one serine residue, makes flagellin composition activation clock sample receptor 5 thus.

153. according to the described method of claim 152, it further comprises at least a antigenic at least a portion.

154. one kind stimulates individual interior immunoreactive method, it comprises this individual step is given in a kind of compositions administration of flagellin composition that it is included as at least a portion of flagellin, wherein, at least one lysine of described flagellin composition is replaced by at least one histidine residues, makes flagellin composition activation clock sample receptor 5 thus.

155. according to the described method of claim 154, it further comprises at least a antigenic at least a portion.

156. one kind stimulates individual interior immunoreactive method, it comprises the compositions administration that a kind of clock sample receptor stimulating agent that is included as at least a portion of clock sample receptor stimulating agent is formed and gives this individual step, wherein, described clock sample receptor stimulating agent is formed the cysteine residues that comprises at least one, be arranged in natural clock sample receptor stimulating agent and locating of cysteine residues do not occur, and make this clock sample receptor stimulating agent form activation clock sample receptor thus.

157. according to the described method of claim 156, it further comprises at least a antigenic at least a portion.

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