CN1887907A

CN1887907A - Recombinant Plasmodium falciparum 175kD erythrocyte conjugated antigen functional region protein and its prepn and use

Info

Publication number: CN1887907A
Application number: CNA2005100272331A
Authority: CN
Inventors: 潘卫庆; 张冬梅
Original assignee: Second Military Medical University SMMU
Current assignee: Second Military Medical University SMMU
Priority date: 2005-06-29
Filing date: 2005-06-29
Publication date: 2007-01-03
Anticipated expiration: 2025-06-29
Also published as: CN100457780C

Abstract

The present invention is one kind of 175 kD recombinant protein (PfEBA175-IIF2) of plasmodium falciparum erythrocyte conjugated antigen functional region, its coding DNA sequence, vector containing the DNA sequence, host cell containing the vector, gene engineering process of preparing the recombinant protein, and the application of the recombinant protein in preparing malaria resisting vaccine and performing preclinical research on plasmodium invasion mechanism. The recombinant protein PfEBA175-IIF2 possesses excellent immunogenicity and good bionomics as well as the function of combining with sialic acid dependent erythrocyte, and can initiate effective plasmodium resisting immune response in immunized individuals.

Description

Recombinant Plasmodium falciparum 175 kD erythrocyte conjugated antigen functional region protein and method for making thereof and purposes

Technical field

The present invention relates to DNA recombinant technology and recombinant vaccine field.More specifically, the present invention relates to a kind of Plasmodium falciparum 175 kD erythrocyte conjugated antigen functional region recombinant protein that contains, the encode dna sequence dna of this recombinant protein, the carrier that contains this dna sequence dna, the host cell that contains this carrier, prepare the method for this recombinant protein with genetically engineered, and this recombinant protein is in the application aspect drug target protein and development malaria vaccine.

Background technology

Malaria is one of the most ancient human transmissible disease, still has a strong impact on human beings'health at present.According to the latest survey of The World Health Organization (WHO), 40% of about world population still is among the malaria threat, is distributed in more than 100 country.There are every year 3 to 500,000,000 people to suffer from malaria at present in the world, wherein about 3,000,000 people's death.Even more serious is that because plasmodium and the drug-fast generation of mosquito matchmaker and diffusion rapidly, malaria not only is not effectively controlled, and the gesture of staging a comeback is arranged on the contrary.Therefore, carry out the plan of whole world containment malaria and become WHO at one of new millennium two priority research areass.

The three approach that mainly contains that people rely in malaria control or expect to obtain to break through: the anti-system of antimalarial drug, malaria vaccine and mosquito matchmaker.But the anti-system of antimalarial drug and mosquito matchmaker is faced with huge difficulty and challenge, and this is because plasmodium and drug-fast generation of mosquito and diffusion.Although there is not the malaria vaccine of using value to come out at present as yet, generally believe that the development malaria vaccine is human control and even the important channel of eliminating malaria.

Studies show that in a large number malaria can realize preventing by developing effective vaccine.Can protect afterwards challenge infection fully as volunteer with the immunity of deactivation sporozoite.Use the plasmodial recombinant antigen immune mouse of mouse for another example, also can protect plasmodial challenge infection of the same race afterwards fully.In addition, the malaria epidemiological study shows that also that die from malaria mainly is the crowd of no malaria immunizing power, as: children and non-malaria district crowd in the malaria district enter the malaria district, and seldom dead because of malaria the adult in malaria district.Inoculate effective vaccine as crowd, make it reach same immunizing power, so just can realize the target of prevention of malaria and reduction malaria mortality ratio with malaria district adult to these no immunizing power.Therefore, the malaria vaccine development has become the hot in nature some problem in the world today.

In malaria vaccine research, there is the research of two candidate vaccines once to be subjected to extensive concern, the one, anti-sporozoite vaccine.This vaccine can suppress sporozoite invasion liver cell.But poor effect in clinical trial afterwards.According to one's analysis, its major cause is that this vaccine only produces anti-sporozoite immunizing power, attacks and survives even there are extremely indivedual sporozoites to escape this vaccine immunity like this, just can invade and breed at liver cell growth, produces enough merozoites and causes that the host is pathogenic.Another is a SPf.66 multivalence synthetic peptide vaccine.This vaccine is one and has only 3 10 peptide epitopes by the hydrophobic amino acid polymer [Patarroyo of 45 peptides of being formed by connecting of being separated by, M.E., et al, induction of protective immunity against experimental infection with malaria suingSynthetic peptides, Nature, 328:629,1987].This vaccine had once been obtained immune protective effect preferably in the douroucouli challenge trial, but failed to obtain the ideal clinical effectiveness afterwards in the test in place of Africa, South East Asia and South America, did not have using value.The reason of this vaccine failure may be that SPf.66 has only 45 peptides, and in short peptide sequence like this, may not have enough t cell epitopes can combine with the individual MHC molecule of majority, causes angtigen presentation to be obstructed.

175kD erythrocyte binding antigen (EBA-175) is plasmodium falciparum synthesizes and be released into blood when schizont breaks when the erythrocytic stage schizogony a kind of soluble proteins.To studies show that of EBA-175, it may be a very important function albumen in the life history of plasmodium falciparum.Yet though once there was the people to cross 175kD erythrocyte binding antigen (EBA-175) at expression in escherichia coli, the EBA-175 that expresses does not but have immunogenicity.In addition, for certain unknown cause, though there is the people to attempt in eukaryotic cells such as yeast, expressing EBA-175, the report of the 175kD erythrocyte binding antigen of successfully not expressing still.Therefore, people think that always EBA-175 can't practice as antimalarial immunogen.

In sum, although carried out nearly 20 years, still do not have the vaccine that using value is arranged so far and come out based on the malaria vaccine research of biotechnology.

Therefore, this area presses for exploitation antimalarial effective immunogen and relevant vaccine.

Summary of the invention

One object of the present invention just provides a kind of new immunogen that can be used for malaria vaccine.Described immunogen is the recombinant protein that contains Plasmodium falciparum 175 kD erythrocyte conjugated antigen functional region (PfEBA175-IIF2).

An object of the present invention is to provide this immunogenic method for making, purposes and contain this immunogenic vaccine composition.

In a first aspect of the present invention, a kind of recombinant protein is provided, it comprises the aminoacid sequence of Plasmodium falciparum 175 kD erythrocyte conjugated antigen functional region IIF2, and has the red corpuscle combined function that sialic acid relies on.More preferably, described recombinant protein has the aminoacid sequence shown in 1-314 position among the SEQ ID NO:2 or the 1-320 position.

In a second aspect of the present invention, provide a kind of isolated DNA molecule, its above-mentioned recombinant protein of the present invention of encoding.More preferably, described dna molecular comprises the nucleotide sequence shown in the SEQ ID NO:1 (comprising 1-960 position or 1-963 position).

In third aspect present invention, the carrier that contains above-mentioned dna molecular is provided, and the host cell that contains described carrier.

In a fourth aspect of the present invention, a kind of method that produces recombinant protein of the present invention is provided, it comprises step:

Under the condition that is fit to the described recombinant protein of expression, cultivate above-mentioned host cell, thereby give expression to described recombinant protein; With separate described recombinant protein.

In a fifth aspect of the present invention, a kind of composition is provided, it contains recombinant protein of the present invention or its coding DNA molecule and pharmaceutically acceptable carrier.

In another preference, described composition is a vaccine composition.

In a sixth aspect of the present invention, a kind of antibody is provided, it is incorporated into recombinant protein of the present invention specifically.

In a seventh aspect of the present invention, the purposes of a kind of recombinant protein of the present invention or Plasmodium falciparum 175 kD erythrocyte conjugated antigen functional region (PfEBA175-IIF2) is provided, it is used to prepare the prevention of malaria vaccine.

Description of drawings

Fig. 1 has shown PfEBA175-IIF2 recombinant protein synoptic diagram and synthetic synoptic diagram.

Wherein, A:PfEBA175-IIF2 recombinant protein synoptic diagram.

Show the position of PfEBA175-IIF2 in total length PfEBA175 molecule among the figure.

SP: signal peptide; I～VI: six districts of total length Pf EBA175 molecule; TM: stride the film district; CYT: cytoplasmic region;

B: the synthetic synoptic diagram of recombinant protein gene Pfeba175-IIf2.

Synthesizing of Pfeba175-IIf2 gene: 959bp Pfeba175-IIf2 gene is divided into 12 oligonucleotide fragments, with asymmetric PCR method proceed step by step gene splicing.Gene 5 ' and 3 ' adds that respectively XhoI and EcoRI site are used for the clone of gene fragment.Each synthetic fragment is cloned in the pBluscript carrier and carries out sequential analysis.

Fig. 2: the agarose of total length Pfeba175-IIf2 gene coagulates electrophorogram.

Electrophoresis showed Pfeba175 synthetic gene band (swimming lane 1).

Fig. 3: immunoblotting detects the electrophorogram and and the erythrocytic association reaction of the expression of PfEBA175-IIF2.

Detect with the on-the-spot human serum in malaria epidemic-stricken area.Wherein, swimming lane 1 is for inducing the supernatant after 72 hours.Swimming lane 2: with the erythrocytic association reaction of rat; Swimming lane 3: with the association reaction of HRBC; Swimming lane 4: the association reaction of the HRBC of handling with neural amino acid enzyme; Swimming lane 5: with the association reaction of the HRBC of trypsin treatment; Swimming lane 6: with the association reaction of rabbit erythrocyte; Swimming lane 7: with the association reaction of mouse red blood cell;

1. swimming lane 8 is the culture supernatant before inducing.

Fig. 4: be the expression that SDS-PAGE measures PfEBA175-IIF2.

Inducing preceding 0hr (swimming lane 2) and inducing back 12 (swimming lanes 3), 24 (swimming lanes 4) and 48 hours (swimming lane 5) to get culture supernatant 15 μ l and carry out that SDS-PAGE separates and coomassie brilliant blue staining.The albumen that occurs clauses and subclauses at the 35KD place.Swimming lane 1 is a molecular weight standard.

Fig. 5: the SDS-PAGE electrophorogram that is the PfEBA175-IIF2 recombinant protein of purifying.

After expressing the supernatant separation,, measure acquisition target protein purity greater than 95% through SPS-PAGE with Ni post and HPLC gluing chromatography two-step purifying.Swimming lane 1:7.5 μ g; Swimming lane 2:15 μ g.

Fig. 6 has shown PfEBA175-IIF2 and malaria epidemic-stricken area patients serum's association reaction situation.

With PfEBA175-IIF2 recombinant protein bag quilt, be one anti-with the malaria epidemic-stricken area patients serum of dilution in 1: 200, carry out ELISA and detect.The result shows, 83% patients serum's (65 example) OD value (0.084) more than the cutoff value.This shows, the antibody at natural PfEBA175-IIF2 that the plasmodial crowd of natural infection produces can be discerned the PfEBA175-IIF2 recombinant protein well, and it is very approaching on conformation further to have reacted PfEBA175-IIF2 recombinant protein and native protein.

Fig. 7: shown the level of measuring the special IgG of immunize rabbit blood-serum P fEBA175-IIF2 with the ELISA method.

Fig. 8: shown the level of measuring the special IgG of immune mouse serum PfEBA175-IIF2 with the ELISA method.Each group is respectively: ◆ IIF2 is immunity separately; ■ IIF2 combined immunization; ▲ PfCP2.9 is immunity separately; * PfCP2.9 combined immunization.

Fig. 9: shown that immune serum suppresses plasmodium growth in vitro efficient-1.

Wherein two groups are respectively: 1 ISA720 adjuvant+Pf EBA175-IIF2 and ISA720 adjuvant+Pf CSP negative control albumen (being 15% serum-concentration).

Figure 10: shown that immune serum suppresses plasmodium growth in vitro efficient-2.

Wherein each group is respectively: the extracorporeal inhibiting rate of special IgG in the extracorporeal inhibiting rate B. immunize rabbit antiserum(antisera) of special IgG in the A. immune monkey antiserum(antisera).

Embodiment

The inventor is extensive studies through going deep into, by to the transformation of the different sections of 175kD erythrocyte binding antigen and to the optimization of expression system, successfully in Yeast system, made first and contain 175kD erythrocyte binding antigen functional zone IIF2 and have immunogenic recombinant protein.Test shows, this recombinant protein can cause immunne response effectively, has the biological characteristicses such as red corpuscle combined function that sialic acid relies on, and is similar to the native conformation of 175kD erythrocyte binding antigen on conformation.Finished the present invention on this basis.

175kD erythrocyte binding antigen (EBA-175) is plasmodium falciparum synthesizes and be released into blood when schizont breaks when the erythrocytic stage schizogony a kind of soluble proteins, is made up of 616 amino-acid residues.

EBA-175 by Camus and Hardley reported first in 1985, this antigen is plasmodium falciparum synthesizes and be released into blood when schizont breaks when the erythrocytic stage schizogony a kind of soluble proteins, this albumen N-end is a signal peptide sequence, contain an intracellular region of striding film district and C-latter end, extracellular region can be divided into 6 zones, wherein II district and VI district be for being rich in the halfcystine district, and what the II district contained two copies is rich in halfcystine district (Figure 1A).

Studies show that, in six kinds of different plasmodium falciparum strain isolateds, all find EBA-175, and all these strain isolateds and EBA-175 peptide 4 antiserum(antisera)s and SAR175 antiserum(antisera) (preparing) produce very consistent positive reaction and show from the douroucouli that infects the CAMP strain isolated.Perhaps, there is not nuance in this prompting between the homophyletic EBA-175 molecule, but the major function territory guards on protein structure and conformation, is extremely similar on biological chemistry and immunological properties.

EBA-175 antigen has two key characters: the one, it can with the sialic acid specific combination of HRBC glycophorin, the 2nd, it can combine with merozoite.The functional domain of receptors bind on existence and the red corpuscle in EBA-175.The II district that the N end of EBA-175 is rich in halfcystine is the mode bonded functional zone that EBA-175 and red corpuscle rely on sialic acid.The II district is made up of two repeated fragment F1 and F2 fragment, and the F2 fragment is itself and red corpuscle bonded zone, and it is identical with total length EBA-175 albumen with red corpuscle bonded mode.

As used herein, term " 175kD erythrocyte binding antigen functional zone II district F2 fragment ", " 175kD erythrocyte binding antigen functional zone IIF2 " or " EBA-175IIF2 " are used interchangeably, and all refer to the F2 fragment in II district, 175kD erythrocyte binding antigen functional zone.This fragments sequence is known, for example the 447-760 amino acids among the GenBank accession number U32207.

As used herein, term " recombinant proteins of 175kD erythrocyte binding antigen functional zone ", " EBA-175IIF2 recombinant protein " etc. are used interchangeably, and all are meant the albumen that the aminoacid sequence by Plasmodium falciparum 175 kD erythrocyte conjugated antigen functional region IIF2 constitutes.In addition, the recombinant protein of 175kD erythrocyte binding antigen functional zone comprises and contains or do not contain signal peptide, and the recombinant protein that contains or do not contain initial methionine.

As used herein, " aminoacid sequence of plasmodium 175kD erythrocyte binding antigen functional zone IIF2 " refers to the aminoacid sequence in recombinant protein of the present invention in the term recombinant protein, this sequence has substantially the same aminoacid sequence with plasmodium EBA-175IIF2 fragment natural or variation, and has the biological characteristicses such as red corpuscle combined function of antigenic activity substantially the same with natural plasmodium EBA-175 and sialic acid dependence.Preferred plasmodium EBA-175IIF2 aminoacid sequence comprises (but being not limited to): second aminoacid sequence that is rich in the halfcystine district in II district in the natural EBA-175 extracellular region, and eliminated this aminoacid sequence of glycosylation site.

In addition, also can add the aminoacid sequence that other do not influence biological characteristicses such as EBA-175IIF2 immunogenicity and red corpuscle combined function at the N of EBA-175IIF2 recombinant protein end or C-terminal.Preferably, the aminoacid sequence of these interpolations helps expressing (as signal peptide), help purifying (as 6xHis sequence, yeast saccharomyces cerevisiae α-factor signal peptide cleavage site (Glu-Lys-Arg)), maybe can promote the immunogenicity (for example sequence of cytokine such as IL-2, GM-CSF) of EBA-175IIF2 recombinant protein.

The dna sequence dna of code book invention recombinant protein, all synthetic.Also available pcr amplification or synthetic method obtain the DNA sequences encoding of EBA-175IIF2, form the dna sequence dna of code book invention recombinant protein.

In order to improve the expression amount of host cell, can transform EBA-175IIF2 recombinant protein encoding sequence, for example adopt the codon of host cell preference, eliminate the sequence that is unfavorable for genetic transcription and translation.In one embodiment of the invention, just adopt the codon of yeast preference, EBA-175IIF2 recombinant protein gene is detected, eliminating is unfavorable for the sequence of genetic transcription and translation in gene, comprise the intron shearing site, transcription termination sequence etc., thus obtained to be beneficial to the encoding sequence of in yeast, expressing.

After the dna sequence dna that has obtained code book invention recombinant protein, it is connected into suitable expression vector, change proper host cell again over to.At last, cultivate the host cell after transforming, obtain new recombinant protein of the present invention by separation and purification.

As used herein, term " carrier " comprises plasmid, clay, expression vector, cloning vector, virus vector etc.

In the present invention, can select various carrier known in the art such as commercially available carrier for use.Such as, select commercially available carrier for use, the nucleotide sequence of then code book being invented new recombinant protein operationally is connected in expression regulation sequence, can form protein expression vector.

As used herein, " operationally being connected in " refers to a kind of like this situation, and promptly some part of linear DNA sequence can influence the activity of same other parts of linear DNA sequence.For example, if signal peptide DNA as precursor expression and participate in the secretion of polypeptide, signal peptide (secretion leader sequence) DNA operationally is connected in polypeptid DNA so; If transcribing of promotor control sequence, it is operationally to be connected in encoding sequence so; When if ribosome bind site is placed in the position that can make its translation, it is operationally to be connected in encoding sequence so.Generally, " operationally being connected in " means adjoining, then means in reading frame adjacent for the secretion leader sequence.

In the present invention, term " host cell " comprises prokaryotic cell prokaryocyte and eukaryotic cell.The example of prokaryotic host cell commonly used comprises intestinal bacteria, Bacillus subtilus etc.Eukaryotic host cell commonly used comprises yeast cell, insect cell and mammalian cell.Preferably, this host cell is an eukaryotic cell, more preferably is yeast cell.

After obtaining transformed host cells, can under the condition that is fit to expression recombinant protein of the present invention, cultivate this cell, thereby give expression to recombinant protein.And then isolate the recombinant protein of expression.

On the other hand, the present invention comprises that also EBA-175IIF2 recombinant protein or its coding DNA are had specific polyclonal antibody and monoclonal antibody, especially monoclonal antibody.Here, " specificity " is meant that antibody capable is incorporated into EBA-175IIF2 recombinant protein or fragment.Preferably, refer to that those can combine with EBA-175IIF2 recombinant protein or fragment but nonrecognition and be incorporated into the antibody of other irrelevant antigen molecule.The present invention also comprise those can with modify or without the EBA-175IIF2 recombinant protein bonded antibody of modified forms.

The present invention not only comprises complete mono-clonal or polyclonal antibody, but also comprises having immunocompetent antibody fragment, as Fab ' or (Fab) ₂Fragment; Heavy chain of antibody; Light chain of antibody; Genetically engineered strand Fv molecule; Or chimeric antibody.

Antibody of the present invention can be prepared by the known various technology of those skilled in that art.For example, the EBA-175IIF2 recombinant protein of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing EBA-175IIF2 recombinant protein or its has antigenic segmental cell and can be used to immune animal and produce antibody.Monoclonal antibody of the present invention can utilize hybridoma technology prepare (see people such as Kohler, Nature256; 495,1975; People such as Kohler, Eur.J.Immunol.6:511,1976; People such as Kohler, Eur.J.Immunol.6:292,1976; People such as Hammerling, In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981).

Available EBA-175IIF2 recombinant protein of the production of polyclonal antibody or polypeptide immune animal, as rabbit, mouse, rat etc.Multiple adjuvant can be used for the enhancing immunity reaction, includes but not limited to freund adjuvant etc.

In another aspect of this invention, provide and contained recombinant protein of the present invention as the combinations of immunogens thing, especially vaccine composition, described composition contains the described recombinant protein of 0.001-99.9wt%, (b) pharmaceutically acceptable carrier and (c) optional adjuvant, wherein each component sum is 100wt%.Vaccine of the present invention can be preventative (being preventing infection) or curative (promptly treating disease after infection).

These vaccines comprise immunity antigen or immunogen, immunogenic polypeptide, albumen or protein fragments or nucleic acid (as Yeast Nucleic Acid or thymus nucleic acid), usually with " pharmaceutically acceptable carrier " combination, these carriers comprise itself does not induce any carrier of generation to the individual deleterious antibody of accepting said composition.Suitable carriers normally big, metabolism macromole slowly, as the virion of protein, polysaccharide, poly(lactic acid), polyglycolic acid, aminoacid polymers, amino acid copolymer, lipid agglutinator (as oil droplet or liposome) and non-activity.These carriers are well known to those of ordinary skill in the art.In addition, these carriers can play immunostimulant (" adjuvant ") effect.

The preferable adjuvant of enhancing composition effect is including, but not limited to (1) aluminium salt (alum), as aluminium hydroxide, aluminum phosphate, Tai-Ace S 150 etc.; (2) ISA720 adjuvant; (3) freund adjuvant etc.

Vaccine composition (comprising antigen, pharmaceutically acceptable carrier and/or adjuvant) contains thinner usually, as water, and salt solution, glycerine, ethanol etc.In addition, complementary material can be present in this class vehicle as wetting agent or emulsifying agent, pH buffer substance etc.In addition, the vaccine composition that comprises immunogenic composition can contain antigen, polypeptide, albumen, protein fragments or the nucleic acid in pharmaceutically acceptable carrier.

More specifically, comprise the vaccine of immunogenic composition, comprise the immunogenic polypeptide of immune significant quantity, and above-mentioned other required component." immune significant quantity " refers to that giving individual amount with a single agent or a continuous agent part is effective to treatment or prevention.This consumption according to the individual healthy state of treat and physiological situation, institute treat the ability of individual classification (as non-human primates etc.), individual immunity system synthetic antibody, required degree of protection, vaccine preparation, treat the doctor assessment of medical conditions, the correlative factor that reaches other decided.Estimate that this consumption will can determine by normal experiment in the scope of relative broad.

Usually, vaccine composition or immunogenic composition can be made the injectable agent, for example liquor or suspension; Also can be made into the solid form that before injection, is fit to allocate into solution or suspension, liquid excipient.But also emulsification or be encapsulated in the liposome of said preparation strengthens adjuvant effect under above-mentioned pharmaceutically acceptable carrier.

Ordinary method is to give immunogenic composition from parenteral (subcutaneous or intramuscular) approach by injection.That other prescription that is fit to other administering mode comprises is oral, suppository and transdermal application etc.Therapeutic dose can be single agent scheme or multi-agent scheme.Vaccine can give together in conjunction with other immunomodulator.

A kind of vaccine mode is a dna vaccination, promptly contains the dna vaccination of the encoding sequence of code book invention recombinant protein.

In an example of the present invention, complete synthesis Plasmodium falciparum 175 kD erythrocyte conjugated antigen functional region gene, and in pichia yeast this antigen of secreting, expressing, its conformation is very near native conformation by analysis.This antigenic immune serum can effectively suppress growth of malaria parasites, and has the biological characteristicses such as red corpuscle combined function that sialic acid relies on.

Major advantage of the present invention is:

(1) EBA-175IIF2 recombinant protein of the present invention, its conformation be very near the native protein conformation, and have the good biological characteristicses such as red corpuscle combined function that sialic acid relies on.

(2) EBA-175IIF2 Recombinant Protein Expression product can be secreted into outside the born of the same parents, and enters no albumen culture supernatant, is convenient to separation and purification, and purity can reach more than 95%.

(3) EBA-175IIF2 Recombinant Protein Expression level height.Shaking bottle expression amount is 20mg/L, and 15L fermentation expression output can reach 300mg/L.

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989 or Chinese patent application CN01105292.9) described in condition, or the condition of advising according to manufacturer.

Embodiment 1

Synthesizing of PfEBA-175IIF2 gene

1.1.Pfeba-175IIf2 the design of complete synthesis gene

The proteic sequence of reorganization EBA-175IIF2 is from the aminoacid sequence of plasmodium falciparum 3D7 strain 175kD erythrocyte binding antigen functional zone EBA-175IIF2.Select the pichia yeast codon usage frequency for use, this sequence is redesigned.Adopt analysis such as computer software " DNA Star ", Omiga to remove any sequence that may be unfavorable for genetic transcription and translation that in this gene order, exists side by side, as transcription termination sequence, intron splice site sequence, the inverted repeats grown etc.In addition, in 5 of newly-designed eba-175IIf2 gene ' and 3 ' end sets up XhoI and EcoRI restriction enzyme site respectively, so that can be connected with expression vector.Identify potential glycosylation site in this antigen sequence, and by glycosylation site amino acid Asn being transferred to Gln (seeing SEQ ID NO:2), thereby this glycosylation site got rid of.

EKREHIDLDD?FSKFGCDKNS?VDTNTKVWEC?KKPYKLSTKD?VCVPPRRQEL 50

CLGNIDRIYD?KNLLMIKEHI?LAIAIYESRI?LKRKYKNKDD?KEVCKIIQKT 100

FADIRDIIGG?TDYWNDLSNR?KLVGKINTNS?NYVHRNKQND?KLFRDEWWKV 150

IKKDVWNVIS?WVFKDKTVCK?EDDIENIPQF?FRWFSEWGDD?YCQDKTKMIE 200

TLKVECKEKP?CEDDNCKRKC?NSYKEWISKK?KEEYNKQAKQ?YQEYQKGNNY 250

KMYSEFKSIK?PEVYLKKYSE?KCSNLNFEDE?FKEELHSDYK?NKCTMCPEVK 300

DVPISIIRNN?EQTSHHHHHH 320

(SEQ?ID?NO：2)

Add the partial sequence of yeast saccharomyces cerevisiae α-factor signal peptide leader sequence simultaneously at 5 of this gene ' end, the codon sequence (nucleotides sequence is classified aaa aga gag gct gaa gct as) that comprises signal peptide cutting identification amino acid Lys-Arg-Glu-Ala-Glu-Ala, make it secreting, expressing, and 3 ' end sets up the sequence of 6 Histidines of coding, so that use the Ni-NTA column purification.Consequent dna sequence dna is made up of 959bp, and names and be eba-175IIf2.Like this, the EBA-175IIF2 recombinant protein is made up of 320 amino acid.Fig. 1 shows the synthetic gene synoptic diagram.

1.2.Pfeba-175IIf2 complete synthesis gene synthetic

Referring to Figure 1B.The eba-175IIf2 synthetic gene sequence is divided into 12 oligonucleotide chains, from 5 ' be eba-175IIf2-1 to eba-175IIf2-12, the lap of 16 to 20 bases of having an appointment between adjacent two oligonucleotide chains to 3 ' successively lives.Adopt gene software Oligo 4.0 to analyze adjacent oligonucleotide interchain lap sequences, be unfavorable for the sequence of oligonucleotide chain splicing, as hairpin structure etc. with eliminating.

With PCR method 12 chains are spliced into double chain DNA molecule.The pcr amplification condition is: 95 ℃ 10 seconds (sex change), 55 ℃ 30 seconds (annealing) and 72 ℃ 1 minute 30 seconds (extension), each 25 circulations produce 959bp total length eba-175IIf2 synthetic gene.The PCR product separates with 1% agarose gel, and reclaims test kit with QIAGEN DNA and separate goal gene (Fig. 2).With XhoI and EcoRI double digestion target gene fragment, the enzyme tangent condition is: in 20 μ l reaction systems, contain 2 μ g target gene fragment, and 1 * enzyme cutting buffering liquid, each 5 unit of XhoI and EcoRI enzyme, 37 ℃ were reacted 1 hour.Endonuclease bamhi is connected with the pBluscript carrier of cutting through same enzyme, and transformed competence colibacillus DH5 α intestinal bacteria.The plasmid DNA of extracting acillin resistance bacterium colony is carried out dna sequence analysis after double digestion is accredited as correct insertion.Wrong base when synthesizing to get rid of.

The plasmid that will contain the faultless synthetic gene of total length changes bacillus coli DH 5 alpha over to, after inoculation culture repeatedly, reclaims this plasmid and carries out sequential analysis, and the result does not have the base mutation of discovery in the eba-175IIf2 gene.Show in these synthetic gene energy stable existence intestinal bacteria.

Pfeba-175IIf2 sequence after the order-checking is shown in SEQ ID NO:1.

For make the Pfeba-175IIf2 gene can be in pichia yeast secreting, expressing, this gene 5 ' end is modified, promptly add the partial sequence of yeast saccharomyces cerevisiae α-factor signal peptide leader sequence, comprise the codon sequence (nucleotides sequence is classified aaa agagag gct gaa gct as) of signal peptide cutting identification amino acid Lys-Arg-Glu-Ala-Glu-Ala at 5 of this gene ' end.In 5 of Pfeba-175IIf2 gene ' and 3 ' end contains XhoI and EcoRI site respectively and is used for this gene clone and goes into expression vector.

Embodiment 2

Pfeba-175IIf2 gene secreting, expressing in pichia yeast

2.1: the structure of expression vector

The Pfeba-175IIf2 gene is inserted into pPIC9 expression plasmid of yeast (purchasing the company in lnvitrogen) by XhoI and EcoRI site.Like this, yeast saccharomyces cerevisiae α-factor signal peptide C-terminal merges on the N of PfEBA-175IIF2 end and this carrier.Because PfEBA-175IIF2 molecule N end contains three the special amino acid Glu-lys-Arg sequences in this signal peptide point of contact.So the target protein that secreting, expressing discharges does not contain signal peptide sequence.

The primary element of pPIC9K carrier is identical with pPIC9, but it has kalamycin resistance gene, and available G418 carries out the screening that high copy inserts son.Like this, will contain in the corresponding site that the Pfeba-175IIf2 fragment cut out and inserted pPIC9K expression plasmid (purchasing the company in lnvitrogen), be built into pPIC9K/Pfeba-175IIf2 with BamHI and SalI.

2.2: transform pichia yeast SMD1168

With the pPIC9K/Pfeba-175IIf2 plasmid linearization, 10 μ g linearizing expression plasmids electricity transforms the SMD1168 pichia yeast with SalI.Transformed bacteria is used the transformant of different concns G418 plate screening multiple copied insertion then earlier with histidine defect plate screening His+ transformant.The genomic dna of the different transformants of extracting is identified with the inner primer PCR of a pair of goal gene, shows that expression plasmid inserts yeast chromosomal.There is the transformant of insertion to carry out following expression study through evaluation.

2.3:PfEBA-175IIF2 express

It is in the substratum of carbon source that transformant is seeded in earlier with glycerine, grows after 24 hours, and what be transferred to is in the substratum of carbon source with methyl alcohol.Carry out abduction delivering.Cleer and peaceful cell precipitation on sampling in per 24 hours, the detection expression product.SDS-PAGE and Xylene Brilliant Cyanine G detected result show: tangible PfEBA-175IIF2 band of expression is arranged at the 35kD place, and this expression band only occurs behind methanol induction, and with the expression time prolongation, its expression product obviously increases (Fig. 4).Carry out immunoblotting with Chinese malaria district malaria disease human serum and detect expression product, the visible 35kD target protein of result trace.

Embodiment 3.

PfEBA-175IIF2 fermentation and purifying

Through optimization research, shake bottle expression level and can reach 20mg/L expression condition.Carry out fermentation expression with the 15L jar.In whole fermentation period, the cell in early stage is exponential growth and rises, and cell density reaches OD ₆₀₀=100.Add vegetative period to glycerine, the cell growth is linearly risen, and cell density reaches OD ₆₀₀=560.But arrived the methanol induction expression phase, the cell overall consistency remains unchanged substantially, and target protein to express be to add back 3-7hr at methyl alcohol to begin, and prolong in time, expressing productive rate improves rapidly, and constantly is secreted in the fermented liquid, high expression level amount can reach 300mg/L.

The purifying of PfEBA-175IIF2 expression product carries out in two steps: the first step is to use the Ni-NTA column purification.Because the PfEBA-175IIF2C end contains 6 * His sequence, thus expression product can be attached on the Ni-NTA affinity column, and with 250mM imidazoles wash-out target protein.Second step was with gel-filtration liquid phase method purifying.Purity of protein through two-step purifying can reach 95% (Fig. 5).

Embodiment 4

Use the PfEBA-175IIF2 immune animal

In this embodiment, the PfEBA-175IIF2 with purifying is an antigen earlier, is the immunological adjuvant immunizing rabbit with ISA720, and experiment is divided into 7 groups, and first group is ISA720+PfEBA-175IIF2; Second group is ISA720+PfCP-2; The 3rd group is ISA720+PfCSP; The 4th group is ISA720+PfEBA-175IIF2+PfCP-2; The 5th group is ISA720+PfEBA-175IIF2+PfCP-2+PfCSP; The 6th group is ISA720+ sex change PfEBA-175IIF2; The 7th group is the contrast of ISA720 adjuvant.Immunity divides to be carried out for 4 times, and each immunity was carried out at the 0th, 20,40 and 61 day respectively.Reaching immunity before each immunity finishes to get hematometry specific antibody level (Fig. 7) about two weeks of back.Concrete grammar is as follows:

PfEBA-175IIF2 antigen mixes by 3: 7 with the ISA720 adjuvant, with syringe pump emulsification extremely evenly.PfEBA-175IIF2 antigen denaturing treatment is carried out by the following method: antigenic solution mixes with solid urea, and making final concentration is 8M, puts 50 ℃ of water-bath 60min, and it is 20mM that adding DTT makes its final concentration, puts 50 ℃ of water-bath 6hr; Adding sodium iodoacetate again, to make its final concentration be 60mM, puts room temperature 60min, finishes the back with 0.1M pH7.5 borate buffer (100mM boric acid, 137mM NaCl transfer pH to 7.5) dialysed overnight.Immunizing dose is every rabbit 200 μ g, is the 1ml volume.Adopt subcutaneous multi-point injection.Four immunization times are respectively at D0, D20, D40 and D61.D33 and D54, D75 get hematometry antibody ELISA and IFA titre after first immunisation

ELISA is undertaken by following program: adopting expression PfEBA-175IIF2 or the full worm albumen of plasmodium falciparum is antigen, every hole package amount is 0.1 μ g, add different dilution antiserum(antisera) 100 μ l, every part of dilute serum sample repeats three holes, after 37 ℃ of reaction 1hr and washing, add ELIAS secondary antibody,, and read 450mM OD value with the colour developing of TMD substrate.Determining positive threshold value with preimmune serum, is the final titre of antiserum positive greater than the extent of dilution of this value.

ELISA result shows that the adjuvant control group does not produce specific antibody, and all the other 6 groups all produce tangible specific antibody (Fig. 7).The highest antibody titers is 1: 1,389,224; And have only 1: 3 with the sero-fast antibody titers of reorganization PfEBA-175IIF2 protein immunization rabbit of sex change, 228.

In addition, be antigen at PfEBA-175IIF2 with purifying, be that immunological adjuvant immunity rhesus monkey and BALB/c mouse also produce tangible specific antibody (Fig. 8) with ISA720.The highest antibody titers is 1: 41,026 (rhesus monkey), 1: 61,435 (BALB/c mouse).

The above results shows that this recombinant protein demonstrates high immunogenicity in mouse, rabbit, rhesus monkey immunization experiment, far above the PfEBA-175IIF2 albumen of sex change.

Embodiment 5

PfEBA-175IIF2 is to plasmodial body outer suppressioning test

Hainan Province is taken from the plasmodium falciparum FCC1/HN strain that is used for this experiment.Cultivation is undertaken by general in the world TragerShi wax candle jar method, and calculates as follows and suppress protozoon growth rate (inhibiting rate)

Concrete grammar is as follows: plasmodium falciparum FCC1/HN strain is carried out vitro culture by Trager and JansenShi method, changes nutrient solution every day, per four days interpolation red corpuscle.The protozoon that is used for inhibition test need pass through synchronous processing, and promptly in experiment preceding 24 hours, the plasmodium that contains each phase mixed with 5% sorbyl alcohol, puts room temperature 30min.Protozoon through this processing mainly only contains the ring bodies protozoon in period.Continue to cultivate 24 hours, the overwhelming majority has been grown schizont again.The protozoon of this moment is mixed with the protozoan infection rate of 2% cell pack and 0.5%.Need to add different amount antiserum(antisera)s by experiment, be mixed with the starting culture of different antiserum(antisera) concentration.With every hole 200 μ l this culture is put in the 96 hole flat boards, cultivated 24hr or after 72 hours, make thin smear film, Giemsa stain, use the microscopic counting parasite rate.

Antiserum Preparation: with sterilizing syringe from the immunizing rabbit heart extracting blood, put in the sterilization centrifuge tube, treat blood coagulation, after blood clot dwindles gradually and overflows serum, centrifugal recovery serum, isolating serum is used for above-mentioned inhibition test after 56 ℃ of 30min deactivations and filtration sterilization.

Remove the sero-fast preparation of IgG: remove antibody in the antiserum(antisera) with the albumin A absorption method.Load an albumin A-Agarose layer folding post, this post is carried out pre-treatment by shop instruction.Add antiserum(antisera) on post, collect filtered solution, cross post repeatedly and fail to see the IgG band on the glue until dying at the SDS-PAGE Xylene Brilliant Cyanine G.The IgG of while elution of bound on post.Body outer suppressioning test result (Fig. 9) ISA720 adjuvant and the antigenic immune serum of PfEBA-175IIF2 can suppress 85% (rhesus monkey serum) and the above protozoacide growth of 82% (rabbit anteserum) after 6.7 times of dilutions, and this effect is to depend on the correct space conformation of PfEBA-175IIF2.

The result is as follows:

(1) rabbit anteserum with ISA720 adjuvant+PfEBA-175IIF2 immunity can suppress the growth of 82% protozoon, and does not only have obvious restraining effect (IR:0%) with the PfCSP negative control group.(Fig. 9)

(2), carry out alkanisation with alkylating agent again and handle with 8M urea and DTT sex change PfEBA-175IIF2 albumen.Lose the growth of malaria parasites effect (IR:0%) that suppresses fully through the PfEBA-175IIF2 of this denaturing treatment and the antiserum(antisera) of ISA720 immunity generation.

(4) remove IgG in each rabbit immune serum with the albumin A post, and measure the inhibition plasmodium falciparum growth in vitro effect of the immune serum of this no IgG.The result shows: the immune serum of ISA720 adjuvant+PfEBA-175IIF2 group, after removing IgG, lose original restraining effect, and its inhibiting rate reduces to 0% from original 82%.

Above inhibition test result shows: the immune serum that (1) PfEBA-175IIF2 produces can effectively suppress plasmodium growth in vitro (＞80% inhibiting rate) after 6.7 times (15% serum content) dilution, and this restraining effect is antibody-mediated, lose original restraining effect because remove the immune serum of antibody, and this restraining effect is that IgG concentration relies on; (2) the ISA720 adjuvant is that existing approved human body is used by the novel adjuvant of France match BIC Corp development.This result shows that ISA720 and PfEBA-175IIF2 combined immunization produce very high antibody horizontal.This has searched out suitable adjuvant for PfEBA-175IIF2 carries out clinical trial as vaccine; (3) immanoprotection action of PfEBA-175IIF2 is to depend on the correct conformation of this antigen, can not induce protection antibody through the antigen of denaturing treatment.Like this, effectively the PfEBA-175IIF2 recombiant vaccine need produce the expression product with native conformation.

In addition, behind reorganization PfEBA175-IIF2 and the PfCP-2.9 combined immunization, be not less than the titre of the independent immunity of two albumen at the ELISA antibody titers of two recombinant proteins: antiserum(antisera) and specific IgG at two recombinant proteins are not answered combined immunization and are weakened (Fig. 7,8) in the ability of vitro inhibition plasmodium falciparum growth.As previously mentioned, because the plasmodium complexity life history, antigen has kind, strain, phase specificity, and exist serious antigenic variation, univalent vaccine be difficult to reach 100% efficient.Therefore, an effective malaria vaccine need have a plurality of antigenic mixing, and the PfEBA175-IIF2 that relates among the present invention carries out the good experimental result that combined immunization obtained with the malaria vaccine candidate antigens PfCP-2.9 that has entered the I clinical trial phase, should be the vaccine development pattern that this a plurality of antigen mixes and brings dawn.

Embodiment 6

Reorganization PfEBA-175IIF2 and erythrocytic association reaction

RPMI1640 nutrient solution with serum-free is thoroughly dialysed to containing the proteic fermentation culture supernatant of reorganization PfEBA-175IIF2 at 4 ℃.The fermentation culture supernatant liquor of dialysing at room temperature respectively with people's intact red cells, the abundant mixing of red corpuscle 60 minutes of HRBC, mouse, rat, rabbit through neural amino acid enzyme or trypsin treatment.The albumen that will be combined on the red corpuscle with 1M NaCl elutes again.Verify reorganization PfEBA-175IIF2 and the erythrocytic situation that combines with the method for SDS-PAGE and Western blot.

The result as shown in Figure 3.Show that reorganization EBA175-IIF2 can combine with intact red cells but and can not combine with the red corpuscle of neuraminidase or trypsin treatment.As seen the EBA175-IIF2 that recombinates has the biological characteristics of the red corpuscle combined function that relies on to the extremely similar sialic acid of native protein.

Embodiment 7

Reorganization PfEBA-175IIF2 and malaria epidemic-stricken area patients serum's association reaction

With PfEBA175-IIF2 recombinant protein bag quilt, be one anti-with the malaria epidemic-stricken area patients serum of dilution in 1: 200, carry out ELISA and detect.

The result as shown in Figure 6.The result shows, 83% patients serum's (65 example) OD value (0.084) more than the cutoff value.This shows, the antibody at natural PfEBA175-IIF2 that the plasmodial crowd of natural infection produces can be discerned the PfEBA175-IIF2 recombinant protein well, and it is very approaching on conformation further to have reacted PfEBA175-IIF2 recombinant protein and native protein.

Discuss

Malaria at present still serious threat human beings'health, the drug-fast generation of plasmodium and rapidly diffusion and anti-sterilant mosquito matchmaker appearance and spread, make the medical treatment of malaria be absorbed in predicament, so the effective malaria vaccine of development has become the task of top priority so that the control malaria is popular.The plasmodium complexity life history, antigen have kind, strain, phase specificity, and have serious antigenic variation, and this brings difficulty to vaccine development, thus the difficulty of malaria vaccine development than people imagined originally much higher.In the life history, have only the erythrocytic stage protozoon to make the people pathogenic even fatal plasmodium, therefore develop sickness rate and the case fatality rate that effective erythrocytic stage malaria vaccine can reduce malaria, significant to the control of malaria; And the invasion red corpuscle is most important to the existence of plasmodium falciparum, plasmodium has only invasion host red corpuscle to survive, therefore this process should be the key link of plasmodium erythrocytic stage in the life history, and plasmodium invasion red corpuscle is a complicated process, be broadly divided into the identification of plasmodium and erythrocytic absorption, plasmodium and red corpuscle combining site and redirect, plasmodium is to several stages such as erythrocytic attack, invasions.Disturb or block this combination and just can block the invasion of plasmodium merozoite, blocking-up plasmodium invasion red corpuscle just should be able to stop the growth of erythrocytic stage protozoacide, should be an importance of malaria vaccine development.Existing experimental evidence shows, the erythrocytic process of plasmodium invasion host is an acceptor and ligand-mediated, and relates to many interactions to receptors ligand, therefore, is that malaria vaccine development institute is essential to the research of different plasmodium ligandins.

Generally speaking, the plasmodium life history and antigenic complicacy make host immune system also very complicated to plasmodial responsing reaction, also increased the difficulty of malaria vaccine development, therefore in the selection of vaccine candidate antigen, more should start with, select the development of malaria vaccine to be had prior meaning with such link life history plasmodium albumen in close relations from the key link of plasmodium the life history.

The invasion red corpuscle is most important to the existence of plasmodium falciparum, and the EBA175-IIF2 albumen with red corpuscle combined function certainly will participate in plasmodium falciparum and invades this important vital movement.The research prompting, this albumen also has stronger immunogenicity, and its immune serum can be in the plasmodial growth of vitro inhibition.In recent years find EBA-175 antigen the pre-erythrocytic stage plasmodium stage also express, make this malaria vaccine candidate antigens enjoy the investigator to pay close attention to.

The inventor is through extensive and deep research, from the complete synthesis Plasmodium falciparum 175 kD erythrocyte conjugated antigen functional region protein gene of dna level and express this recombinant protein.Found that this recombinant protein can cause immunne response effectively, and have the biological characteristicses such as red corpuscle combined function that sialic acid relies on.

In addition, because the plasmodium complexity life history, antigen has kind, strain, phase specificity, and exist serious antigenic variation, univalent vaccine be difficult to reach 100% efficient.Therefore, an effective malaria vaccine need have a plurality of antigenic mixing, and the PfEBA175-IIF2 that relates among the present invention carries out the good experimental result that combined immunization obtained with the malaria vaccine candidate antigens PfCP-2.9 that enters the I clinical trial phase, for the exploitation polyvalent vaccine brings dawn.

All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.

Sequence table

＜110〉Second Military Medical University, PLA

＜120〉recombinant Plasmodium falciparum 175 kD erythrocyte conjugated antigen functional region protein and method for making thereof and purposes

<130>054501

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<211>963

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(963)

＜223〉encoding sequence of 175kD erythrocyte binding antigen functional zone IIF2 recombinant protein

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gaaaagagag?agcatattga?tttggacgat?ttttctaaat?tcggttgtga?caagaactcc 60

gttgatacta?atacgaaagt?ctgggaatgc?aagaaaccat?acaagttgtc?taccaaagac 120

gtttgtgtcc?ctccaagacg?tcaagaattg?tgtcttggca?acattgatag?aatctacgac 180

aagaacttgc?tgatgattaa?agagcacatc?ttggctattg?ccatctatga?atctaggatt 240

cttaagcgta?aatacaagaa?caaagatgac?aaggaggttt?gtaaaatcat?tcaaaagact 300

tttgctgata?tcagagacat?tatcggtgga?acagattact?ggaacgactt?gtctaatcgt 360

aaactggttg?gtaagattaa?cactaattcc?aactacgtcc?atagaaataa?acaaaacgat 20

aagttgttta?gggacgaatg?gtggaaagtt?attaagaaag?atgtctggaa?cgttatctct 80

tgggtgttca?aggacaaaac?tgtttgtaag?gaggatgaca?ttgaaaacat?cccacaattt 540

ttcagatggt?tttccgagtg?gggtgatgac?tactgtcagg?ataaaactaa?gatgattgaa 600

accttgaaag?ttgagtgcaa?ggaaaaacca?tgtgaggacg?ataactgcaa?gagaaaatgt 660

aattcttaca?aggaatggat?ttccaaaaag?aaagaggaat?ataacaagca?agcaaaacag 720

taccaagagt?atcagaaggg?taataactac?aaaatgtatt?ctgaatttaa?gtcaattaaa 780

ccagaggttt?acttgaagaa?atattctgaa?aagtgttcca?atcttaactt?cgaggatgaa 840

tttaaggagg?aattgcattc?tgactacaag?aacaaatgta?ctatgtgccc?agaggttaag 900

gatgtcccta?tttcaatcat?tagaaataac?gaacaaacat?ctcaccatca?ccatcaccat 960

taa 963

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<211>320

<212>PRT

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(320)

＜223〉175kD erythrocyte binding antigen functional zone IIF2 recombinant protein

<400>2

Glu?Lys?Arg?Glu?His?Ile?Asp?Leu?Asp?Asp?Phe?Ser?Lys?Phe?Gly?Cys

1 5 10 15

Asp?Lys?Asn?Ser?Val?Asp?Thr?Asn?Thr?Lys?Val?Trp?Glu?Cys?Lys?Lys

20 25 30

Pro?Tyr?Lys?Leu?Ser?Thr?Lys?Asp?Val?Cys?Val?Pro?Pro?Arg?Arg?Gln

35 40 45

Glu?Leu?Cys?Leu?Gly?Asn?Ile?Asp?Arg?Ile?Tyr?Asp?Lys?Asn?Leu?Leu

50 55 60

Met?Ile?Lys?Glu?His?Ile?Leu?Ala?Ile?Ala?Ile?Tyr?Glu?Ser?Arg?Ile

65 70 75 80

Leu?Lys?Arg?Lys?Tyr?Lys?Asn?Lys?Asp?Asp?Lys?Glu?Val?Cys?Lys?Ile

85 90 95

Ile?Gln?Lys?Thr?Phe?Ala?Asp?Ile?Arg?Asp?Ile?Ile?Gly?Gly?Thr?Asp

100 105 110

Tyr?Trp?Asn?Asp?Leu?Ser?Asn?Arg?Lys?Leu?Val?Gly?Lys?Ile?Asn?Thr

115 120 125

Asn?Ser?Asn?Tyr?Val?His?Arg?Asn?Lys?Gln?Asn?Asp?Lys?Leu?Phe?Arg

130 135 140

Asp?Glu?Trp?Trp?Lys?Val?Ile?Lys?Lys?Asp?Val?Trp?Asn?Val?Ile?Ser

145 150 155 160

Trp?Val?Phe?Lys?Asp?Lys?Thr?Val?Cys?Lys?Glu?Asp?Asp?Ile?Glu?Asn

165 170 175

Ile?Pro?Gln?Phe?Phe?Arg?Trp?Phe?Ser?Glu?Trp?Gly?Asp?Asp?Tyr?Cys

180 185 190

Gln?Asp?Lys?Thr?Lys?Met?Ile?Glu?Thr?Leu?Lys?Val?Glu?Cys?Lys?Glu

195 200 205

Lys?Pro?Cys?Glu?Asp?Asp?Asn?Cys?Lys?Arg?Lys?Cys?Asn?Ser?Tyr?Lys

210 215 220

Glu?Trp?Ile?Ser?Lys?Lys?Lys?Glu?Glu?Tyr?Asn?Lys?Gln?Ala?Lys?Gln

225 230 235 240

Tyr?Gln?Glu?Tyr?Gln?Lys?Gly?Asn?Asn?Tyr?Lys?Met?Tyr?Ser?Glu?Phe

245 250 255

Lys?Ser?Ile?Lys?Pro?Glu?Val?Tyr?Leu?Lys?Lys?Tyr?Ser?Glu?Lys?Cys

260 265 270

Ser?Asn?Leu?Asn?Phe?Glu?Asp?Glu?Phe?Lys?Glu?Glu?Leu?His?Ser?Asp

275 280 285

Tyr?Lys?Asn?Lys?Cys?Thr?Met?Cys?Pro?Glu?Val?Lys?Asp?Val?Pro?Ile

290 295 300

Ser?Ile?Ile?Arg?Asn?Asn?Glu?Gln?Thr?Ser?His?His?His?His?His?His

305 310 315 320

Claims

1. a recombinant protein is characterized in that, it comprises the aminoacid sequence of Plasmodium falciparum 175 kD erythrocyte conjugated antigen functional region IIF2, and has the red corpuscle combined function that sialic acid relies on.

2. recombinant protein as claimed in claim 1 is characterized in that, described recombinant protein has the aminoacid sequence shown in 1-314 position among the SEQ IDNO:2 or the 1-320 position.

3. an isolated DNA molecule is characterized in that, the described recombinant protein of its coding claim 1.

4. dna molecular as claimed in claim 3 is characterized in that, it has the nucleotide sequence shown in the SEQ ID NO:1.

5. a carrier is characterized in that, it contains the described dna molecular of claim 3.

6. a host cell is characterized in that, it contains the described carrier of claim 5.

7. a method that produces the described recombinant protein of claim 1 is characterized in that, comprises step:

Being fit to express under the condition of described recombinant protein, cultivate the described host cell of claim 6, thereby give expression to described recombinant protein;

Separate described recombinant protein.

9. a composition is characterized in that, it contains the described recombinant protein of claim 1 or described dna molecular of claim 3 and pharmaceutically acceptable carrier.

10. composition as claimed in claim 9 is characterized in that described composition is a vaccine composition.