CN105175550B - A kind of synthetic peptide vaccine for resisting porcine circovirus and application thereof - Google Patents
A kind of synthetic peptide vaccine for resisting porcine circovirus and application thereof Download PDFInfo
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Abstract
The present invention provides a kind of polypeptides, and it includes the amino acid sequences of T1-B-T2 or T2-B-T1 configuration arrangement, wherein T1 is the SEQ ID NO:1 after modification；B is SEQ ID NO:5；And T2 is SEQ ID NO:2 after modification and the polypeptide is also in preparation for the purposes in the preventative vaccine of resisting porcine circovirus, therapeutic vaccine.The polypeptide can be used for synthetic peptide or other types of vaccine, and the immunogenicity of vaccine can be improved.
Description
Technical field
The present invention relates to field of biological product, in particular to a kind of synthetic peptide vaccine for resisting porcine circovirus and application thereof.
Background technique
Antibody plays an important role in anti-infectious immunity.Antibody is directly combined with pathogen, prevents pathogenic infection
Normal cell；Antibody can also be acted on bacteriotoxin, block detoxifying function in target cell.In addition, antibody can also be adsorbed in
Pathogen surface, thus activating complement mediate approach come crack pathogen or promote phagocyte, such as macrophage, thermophilic center
The phagocytosis of granulocyte and natural killer it is cell-mediated ADCC effect.In addition, antibody, especially IgG, can also wear
It crosses placenta materna and enters the circulatory system of fetus, to protect infection of the fetus at birth initial stage from pathogen.Because of antibody
Powerful anti-infectious function is understood that the vaccine for man Vaccine effectiveness of nearly all listing is all related to antibody level
(Vaccines,6th edition,S.A.Plotkin)。
Antibody can resist pathogen invasion to shield to body, how improve the immunogenicity of vaccine to enhance
The response level of antibody becomes the critical problem of vaccine research and development field.The secretory cell of antibody is known as thick liquid cell,
From the B cell of activation, especially in lymphoid tissue centrum germinativum B cell (Germinal Center B cells,
GC B).GC B cell differentiation plasmablast needs the lasting stimulation of antigen.Adjuvant component in inactivated vaccine or subunit vaccine
It is largely to continue antigenic stimulus by being sustained antigen to provide to GC B cell, it is promoted to be divided into secretory antibody
Thick liquid cell.In addition to antigenic stimulus, " help " of cd4 t cell that GC B cell also needs to activate, homology (cognate) is
Plasmablast can be broken up.The cd4 t cell of activation is divided into the function subclass different with phenotype under different immune microenvironments,
Including TH1, TH2 and TH17 etc., these activation cd4 t cells be commonly known as helper T lymphocyte (helper T cell,
Th).Wherein TH1 and TH2 has the ability of " help " B cell differentiation plasmablast.
It is newest the study found that the professional helper T lymphocyte for providing B cell " helps " is one different from TH1 and TH2
Novel subclass.Because the subclass expresses chemokine receptors CXCR5 in cell surface height, by the recruitment of Chemokines CC XCL13
Afterwards, migrate to B cell follicular regions and referred to as folliculus helper T lymphocyte (Follicular helper T cell).It is filtering
In bleb district, the Tfh cell of activation provides " help " signals such as CD40L, ICOS, IL-21 to GC B cell, it is ensured that GC B cell
It survives and it is promoted to be divided into thick liquid cell.And " help " of B cell Tfh cell to be obtained, it is necessary to which the TCR of Tfh is identifiable
Epitope peptide fragment is placed on MHC II, and is shown and combined in cell surface for TCR to activate Tfh cell.To sum up, B cell is divided into
The thick liquid cell of secretory antibody successively needs three steps: 1) B cell is special using B-cell receptor (B cell receptor, BCR)
It identifies anisotropicly, in conjunction with antigen, to activate BCR signal path, B cell is made to be in the state of activation, be subsequent cell proliferation
It prepares with plasma cell differentiation；2) after antigen is in conjunction with BCR, B cell is swallowed in a manner of endocytosis (Endocytosis) and is formed
Body (Endosomes) inside to be bitten, is bitten under body low ph condition inside, antigen is degraded, is truncated into the small peptide of 13-25 amino acid, this
A little small peptides are placed on the MHC II molecule of B cell, and are transported to cell surface, are identified for the TCR of Tfh.3) TCR of Tfh
After the antigen for identifying B cell MHC II class molecule submission, through the costimulatory molecule CD28 on film in conjunction with the B7 molecule of B cell
And activate, the Tfh cell upregulation of activation expresses CD40L, which further stimulates B cell in conjunction with B cell membrane receptor CD40,
Activate B cell completely.So induction includes at least two parts compared with the antigen of High antibody level, one is for B cell
In addition the antigenic component of BCR identification is then the t cell epitope that can be identified by TCR, Neither of the two can be dispensed.
All karyocyte surfaces are all distributed MHC class Ⅰmolecule, and MHC II class molecule is distributed mainly on professional antigen and passs
In cell membrane, such as B cell, Dendritic Cells, macrophage.MHC II class molecule is with certain affinity in conjunction with Antigenic Peptide
For compound (pMHC) and submission is in cell surface, and Tfh cell is swashed using the identification of TCR specificity, in conjunction with the compound
It is living.It threatens the Pathogen category of the mankind various, and makes a variation often, thus the high polymorphism of mhc gene and polygenes are very big
Individual is extended to the range of Antigenic Peptide submission, improves individual to the resilience of adverse environment, is conducive to maintain population existence
With continuity.Current research discovery, Antigenic Peptide and the affinity of MHC II class molecule are higher, and the incorporation range of the two is wider, and TCR knows
Not, stronger in conjunction with the ability of pMHC, then generate that responsiveness Tfh cell mass is more, and the immune efficacy of generation is stronger.Therefore, it grinds
The t cell epitope Antigenic Peptide that a kind of pair of MHC II class molecule has universal affinity is made, the great class of current vaccines development is become
Topic.
The experiment of early stage is it has been proved that the generation of antibody needs " help " of cd4 t cell.Based on this, usually B
Cell epitope or the haptens ingredient covalent coupling of other chemical small molecule classes are to carrier protein, on KLH, BSA, OVA, these
Carrier protein is after BCR is identified and combines antigen, with target antigen by endocytosis to B cell, to be processed, be showed in
On MHC II, then submission activates cd4 t cell to cd4 t cell, and corresponding B cell is obtaining activation cd4 t cell help
Afterwards, differentiation plasmablast generates antibody.In addition, in field of vaccinology, usually by the combination of B cell epitope and cd4 t cell epitope
Object makes B cell generate antibody with the help of cd4 t cell as antigen inoculation body, to play the role of immunoprotection.
Although animal has been listed with the aftosa synthetic peptide vaccine comprising cd4 t cell epitope and B cell epitope, such synthetic peptide
Vaccine is most of also in experimental stage, and immune effect need to be discussed.So far, not yet on someone's synthetic peptide vaccine
City, an important reason are still in the primary stage to the understanding of Tfh cell and B cell correlation, designed synthesis
The quality and quantity of the cd4 t cell (i.e. Tfh) for the B cell that peptide vaccine stimulation can help is to be improved.
Summary of the invention
An aspect of of the present present invention provides a kind of polypeptide, and it includes the amino acid sequences of T1-B-T2 or T2-B-T1 configuration arrangement
Column,
Wherein, T1 is the SEQ ID NO:1 after modification；
B is SEQ ID NO:5；
T2 is the SEQ ID NO:2 after modification.
T1, B or T2 can be by intervening sequences come functional connection, and wherein intervening sequence, which can be, any does not influence T1, B
Or the amino acid sequence of T2 structure or function.In one embodiment of the invention, intervening sequence is ε K.
In an embodiment of the present invention, the modification includes one for replacing, adding and/or lacking in original cell epitope
A or multiple amino acid, and the cell epitope after modification has identical immune function with the original cell epitope.
In representative embodiment of the invention, the modification of the SEQ ID NO:1 may include following one or more
Modification:
(1) the 1st amino acids Serine is hydrophobic amino acid；
(2) the 2nd amino acids glutamic acid are replaced into positively charged amino acid；
(3) the 12nd amino acids glutamic acid are replaced into hydrophobic amino acid；With
(4) the 13rd amino acids glycine are replaced into hydrophobic amino acid.
In representative embodiment of the invention, the modification of the SEQ ID NO:2 includes following one or more modifications:
(1) the 1st amino acids proline is replaced into hydrophobic amino acid；
(2) the 3rd amino acids tyrosine substitutions are other aromatic amino acids；
(3) the 6th amino acids glutamine are replaced into hydrophobic amino acid；
(4) the 7th amino acids asparagines are replaced into positively charged or aromatic amino acid；With
(5) the 13rd amino acids threonines are replaced into hydrophobic amino acid.
In one particular embodiment of the present invention, T1 epitope includes amino acid sequence shown in SEQ ID NO:3.
In one particular embodiment of the present invention, T2 epitope includes amino acid sequence shown in SEQ ID NO:4.
Wherein, the combination of above-mentioned epitope by it is artificial synthesized, by protokaryon or eukaryotic expression or pass through genetic recombination skill
Art is embedded in pathogen and obtains.
In another aspect of the present invention, a kind of isolated polynucleotides are provided, it includes one in being selected from the group
Kind:
A) polypeptide as elucidated before is encoded；Or
B) polynucleotides complementary with polynucleotides a) or stingent hybridization.
In another aspect of the present invention, a kind of vaccine is provided, it includes at least one mentioned-above polypeptides.
In the present invention, vaccine of the invention also includes medicine or veterinarily acceptable delivery vector or adjuvant.
In an embodiment of the present invention, route of vaccine administration is selected from intraocular, intranasal, intramuscular, injection, oral, intraperitoneal, skin
Under, local, intradermal and transdermal delivery.
In one aspect of the invention, a kind of vaccine adjuvant is provided, it includes at least one mentioned-above polypeptides.
In addition, polypeptide of the invention is in preparation for the use in the preventative vaccine of resisting porcine circovirus, therapeutic vaccine
On the way.And isolated polypeptide according to the present invention is also within the scope of the present invention for producing the purposes of polynucleotides.
In the present invention, polypeptide of the invention is preparing the purposes in monoclonal or polyclonal antibody.
In the present invention, polynucleotides of the invention are preparing the purposes in polypeptide.
In the present invention, vaccine of the invention is used for preventative vaccine, the therapeutic vaccine of resisting porcine circovirus in preparation
In purposes.
In the present invention, the preventative vaccine, therapeutic of vaccine adjuvant of the invention in preparation for resisting porcine circovirus
Purposes in vaccine.
Beneficial effects of the present invention
By embodiment, inventor has found that T1-B-T2 induction body generates the ability of antibody than single T1-B or T2-B
Or B high 5-10 times, this discovery illustrates that combined sequence of the invention can stimulate body to generate stronger Tfh response, prior
It is to enhance the responsibility of Tfh corresponding antibody can significantly be promoted to generate.It is of the present invention to be produced comprising epitope combination
Object can be widely used for the preventative vaccine of anti-pathogen infection or tumour or autoimmune disease, therapeutic vaccine, with
Induction generates high-caliber antibody；This combined sequence can also be used for producing polyclonal or monoclonal antibody, for diagnose, treat with
And scientific research.Because the present invention is artificial synthetic product, have the characteristics that definite ingredients, quality controllable, target understand, for synthesizing
Peptide vaccine has stronger advantage.
The present invention will be described in more detail with reference to the accompanying drawing.In from detailed description below, of the invention is above-mentioned
Aspect and other aspects of the present invention will be apparent.
Detailed description of the invention
Fig. 1 shows the result of serum diluting multiple after PCV vaccine immune mouse.
Fig. 2 shows the frequencies of different cd4 t cells and B cell epitope combination induction Tfh.
Fig. 3 shows the quantity of different cd4 t cells and B cell epitope combination induction Tfh.
Specific embodiment
Unless stated otherwise, term of the invention has meaning commonly used in the art.
Epitope can be classified as B cell type, T cell type or both B cell type and T cell type, caused by depending on them
The type of immune response.The definition of B cell or t cell epitope is not specific；For example, a kind of peptide epitopes being capable of induction of antibodies production
It is raw, but the epitope can have a kind of sequence simultaneously, and which can be incorporated into people's HLA molecule, so that it is accessible to CTL,
It therefore is that double B cells and T cell are classified for the defined epitope." t helper cell " or " Tfh " is stimulated by specific antigen
When release promote B cell and killer T cell activation and function cell factor any T cell.
" amino acid " refers to compound, and with amino group and carboxylic acid group, 1, the 2- preferably on carbon skeleton is taken
In generation, 1,3- replaces or 1,4- substitute mode.A-amino acid is most preferred, and 20 including finding in albumen kinds of native aminos
The ammonia that sour (they are l-amino acids, in addition to glycine), corresponding D- amino acid, corresponding N- methylamino acid, side chain are modified
Base acid, do not found in albumen biosynthesis acquisition amino acid (such as 4- hydroxy-proline, 5- hydroxyl-lysine,
Citrulling, ornithine, canavanine, djenkolic acid, beta-cyano alanine) and the derivative a-amino acid of synthesis (such as amino
Isobutyric acid, nor-leucine, norvaline, homocysteine and homoserine).Alanine and y-aminobutyric acid are 1 respectively,
3- amino acid and Isosorbide-5-Nitrae-amino acid example, and many other amino acid are well known in the art.The structures such as statine sample
The structures objects such as object (dipeptides including two amino acid, wherein CONH key is replaced by CHOH), hydroxy ethylene (including two amino acid
Dipeptides, wherein CONH key is by CHOHCH2Displacement), the structures object such as amide of reduction (dipeptides including two amino acid, wherein
CONH key is by CH2The displacement of NH key) and the structures object such as thioamides (dipeptides including two amino acid, wherein CONH key is by CSNH key
Displacement) it is also the useful amino acid residue of the present invention.It is available commercial for amino acid of the invention or can passes through routine
The amino acid that synthetic method obtains.
" displacement " refers to by different amino acid or nucleotide subsitution one or more amino acid or nucleotide.
" missing " refers to the missing of one or more amino acid or nucleotide in amino acid sequence or nucleotide sequence.
" insertion " or " addition " refer to the change in amino acid sequence or nucleotide sequence cause with it is naturally occurring or change
Molecule before change is compared, the increase of one or more amino acid or nucleotide.
" one or more amino acid are deleted and/or added in displacement " then refers to, is mutated using well known to kunkel method Kunkel etc.
The amino of the number for the degree that the facture of nucleic acid or polypeptide is replaced, deletion and/or addition can replace, is deleted and/or added
Acid or nucleotide.Above-mentioned mutation is not limited to using known fixed-point mutation method and the mutation of human-induced, is also possible to natural
Obtained from the mutation of existing nucleic acid or protein is isolated and purified.The mutation of amino acid can wrap containing one or more
A amino acid residue is the amino acid that conformation is D- type, rare amino acid existing for nature or manually modified amino acid, this
A little amino acid, which can be, may not be by genetic codon coding.Similar, induction nucleic acid mutates, can be with
It also may include the nucleotide with modification including the naturally occurring nucleotide of nature.
The conservative substitution of amino acid well known by persons skilled in the art is within the scope of the invention.Conserved amino acid substitution
Including an amino acid with another with same type functional group or side chain such as aliphatic, aromatics, positively charged, electronegative
Amino acid replacement.These substitutions can be enhanced oral administration biaavailability, penetrate into central nervous system, target specific cells group
Body etc..As known to those skilled in the art, change, increase or the amino for deleting single amino acids or small percentage in coded sequence
Individual substitutions, deletion or the increase to peptide, polypeptide or protein sequence of acid are " conservative modification variants ", result in ammonia wherein changing
Amino acid as base acid chemical classes substitutes.For example, the following six groups amino acid respectively contained for guarding substitution each other: 1) third
Propylhomoserin (A), serine (S), threonine (T)；2) aspartic acid (D), glutamic acid (E)；3) asparagine (N), glutamine
(Q)；4) arginine (R), lysine (K)；5) isoleucine (I), leucine (L), methionine (M), valine (V)；And
6) phenylalanine (F), tyrosine (Y), tryptophan (W).The conservative alternative of functionally similar amino acid is provided in ability
Be in domain it is well known, other any of alternatives are all possible.
" hydrophobic amino acid " refers to that side chain has the amino acid of high hydrophobicity, such as methionine (M), tryptophan (W), junket
Propylhomoserin (Y), phenylalanine (F), valine (V), leucine (L), isoleucine (I), proline (P) and alanine (A).
" aromatic amino acid " refers to the amino acid containing aromatic rings, such as tyrosine (Y), phenylalanine (F), tryptophan
(W) and thyroxine.
" positively charged amino acid " refer to side chain have positive charge amino acid, such as lysine (K), arginine (R) and
Histidine (H).
" polypeptide " or " amino acid sequence " refers to peptide, oligopeptides, polypeptide or protein and its Partial Fragment, therebetween with peptide bond phase
The amino acid of connection.When " amino acid sequence " in the present invention is related to a kind of amino acid sequence of naturally occurring protein molecule
When, this " polypeptide " or " protein " is not meant to for amino acid sequence to be limited to relevant to the protein molecule complete
Natural acid sequence.Amino acid sequence of the invention can contain additional peptide.As additional peptide, such as polyhistidine mark
For the peptide for signing the Epitope tags such as (His-tag) or Myc, FLAG.
Peptide analogues and peptide mimics are intended to be included within the scope of the present invention, additionally including peptide of the invention salt and
Ester.Peptide analogues according to the present invention optionally include at least one unnatural amino acid and/or C-terminal or N-terminal at least
One blocking groups.The salt of peptide of the invention is physiologically acceptable organic salt and inorganic salts." analog " appropriate
Design can use area of computer aided.
" peptide mimics " refer to peptide according to the present invention to include at least one non-peptide bond such as urea bond, carbamate
Key, sulphonyl amine key, this mode of hydrazine key or any other covalent bond are modified.The design of " peptide mimics " appropriate can be used
Computer assists.
The salt and ester of peptide of the invention are included within the scope of the invention.The salt of peptide of the invention is physiologically acceptable
Organic salt and inorganic salts.The functional derivative of peptide of the invention includes can be by manner known in the art by as residue
On side chain or N or C-terminal group existing for functional group's preparation derivative, and be included in the invention, as long as they keep
Pharmaceutically acceptable, i.e., they do not destroy the activity of peptide and do not assign the composition containing it with toxicity.These derivatives are for example
Aliphatic ester including carboxyl, by the amide of carboxyl caused by being reacted with ammonia or primary amine or secondary amine, by with acyl group portion
Divide the N- acyl derivative of the free amine group of (such as alkanoyl or carbocyclic aroyl) amino acid residue that reaction is formed or passes through
The O- acyl derivative of the free hydroxyl group to be formed (such as seryl- or threonyl residues) is reacted with acyl moiety.
" polynucleotides ", " nucleic acid sequence " or " base sequence " refer to nucleotide, oligonucleotides or polynucleotides and its piece
Section or part.Nucleic acid of the invention can be in the form of RNA (for example, mRNA) or the form of DNA is (for example, cDNA or genome
DNA) exist.DNA can be double-strand, be also possible to single-stranded.Single stranded DNA or RNA can be coding strand (sense strand) or non-coding
It is any in chain (antisense strand).In addition, polynucleotides of the invention can also be in its 5 ' end or 3 ' end fusion code tag labels
The polynucleotides of (sequence label or marker sequence).They can be synthesis or obtaining from natural origin (for example, dividing
From and/or purifying), may include natural, non-natural or modified nucleotide, and it may include naturally
, it is non-natural or change nucleotide between key, such as phosphoramidic acid ester bond or phosphorothioate bond, be used to replace not
Existing phosphodiester bond between the oligonucleotides nucleotide of modification.
" separation " word refers to substance from its original environment (if for example, spontaneous just refer to its native annulus
Border) it separates.Such as it is exactly not to be separated that a spontaneous polynucleotides or polypeptide, which are present in live animal,
Come, and same polynucleotides or polypeptide are separately exactly to separate with some or all substances coexisted therewith in natural system
's.Such polynucleotides or polypeptide can be a part of a certain carrier, be also possible to a part of a certain composition.Since
Carrier and composition are not the ingredients of its natural surroundings, they are still separation.
" nucleic acid hybridization " is well known in the art (see, e.g., Sambrook etc., Molecular Cloning:A
Laboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory, 2001).In general, temperature is higher,
Salinity is lower, then preciseness becomes higher (being difficult to hybridize), so as to obtain more identical polynucleotides.Suitable is miscellaneous
Hand over temperature different according to the length of base sequence or its base sequence.In addition, the invention further relates to hybridize under " stringent condition ".
In the present invention, " stringent condition " refers to, compared with the hybridization and elution under low ionic strength and higher temperature, for example, 42 DEG C of item
Under part, (50% formamide, 5 × SSC (150mM NaCl, 15mM trisodium citrate), the sodium phosphate (pH 7.6) of 50mM, 5 ×
The denatured sheared salmon sperm dna of Denhardt solution, 10% dextran sulfate and 20 μ g/mL) in be incubated overnight, then exist
It is eluted under the conditions of 65 DEG C with 0.1 × SSC.
Vaccine of the invention includes polypeptide or recombination fusion protein and optional adjuvant comprising polypeptide.The vaccine can be with
It is prepared for applying in one of a number of different fashions.An embodiment according to the present invention, the vaccine intranasal administration.Institute
Lymphoid tissue can be applied in any convenient manner by stating vaccine preparation.It is preferable, however, that being applied to it as liquid stream or drop
On the wall of nasal passage.Intranasal compositions can for example be formulated as nasal drop, spray in liquid form, or be suitable for sucking,
It is formulated as pulvis, creme or emulsion.The composition can contain multiple additives, such as adjuvant, excipient, stabilizer, buffering
Agent or preservative.
For simple application, the vaccine is preferably suitable for polypeptide described in the formal distribution of nasal drop or aerosol or again
It is supplied in the container of group fusion protein.In certain preferred embodiments, the vaccine is prepared for mucosal delivery, especially nose
Portion delivers (Arnon etc., Biologicals (biological products) .2001；29(3-4):237-42；Ben-Yedidia etc., Int
Immunol. (Interaational) 1999；11(7):1043-51).
In one embodiment of the invention, by spraying, aerosol or eye drops can apply through eye.It can be by vaccine
It is formulated as liquid or dry powder, to apply as aerosol, spray or eye drops.It is applied as aerosol, eye drops or spray
Composition may include the excipient of one or more types being generally comprised in such composition, such as preservative, viscous
Regulator, tension regulator, tear blocking agent, buffer, stabilizer, carrier, adjuvant etc. are spent, to prepare the system applied through eye
Agent.
In one embodiment of the invention, it is oral for applying, and the vaccine can be mentioned for example in the form of tablet
For, or wrap up in gelatine capsule or microcapsules.Also in yet another embodiment, the vaccine is prepared for parenterally
Application.In some embodiments, the vaccine is prepared for large-scale inoculation, for example, with jet injector or disposable use
Cylindrantherae is used together.Also according to another embodiment, the application is intramuscular.It is described to apply also according to another embodiment
With being intradermal.The needle for being specially designed for intradermal investment vaccine is well known in the art, such as is especially disclosed in the U.S.
In the patent No. 6,843,781 and 7,250,036.According to other embodiments, the application is carried out with needleless injector.
The preparation of these forms is common sense to those skilled in the art.
Delivery vector includes liposome, microparticle, nano particle etc..Liposome is provided for antigen delivery and presentation
Another delivery system.The bilayer vesicle that liposome is made of the phosphatide and other sterols that surround a usual aqueous center,
Antigen or other products can be encapsulated in the aqueous center.Liposome structure be height multiplicity, many types be from
In the range of about 25nm to about 500 μm of nanoscale to micron order.It has been found that liposome can effectively deliver therapeutic agent to skin
And mucomembranous surface.Liposome can be further modified, for for example being targeted by the way that specific antibody is incorporated into skin covering of the surface
Delivering, or it is changed to encapsulating bacterium, virus or helminth.The mean survival time or half-life period of complete liposome structure
It can extend when including certain polymer such as polyethylene glycol, allow extended release in vivo.Liposome can be single layer
Or multilayer.Microparticle and nano particle use small biodegradable sphere, it is used as the reservoir of vaccine delivery.Polymer
It is that they are extremely safe, and are eaten by the U.S. that it is more than the major advantage of the adjuvant of other Reservoir effects that microballoon, which has,
Product and drugs administration approved are as suitable suture for human medical and as biodegradable drug delivery system
System.The rate of copolymer hydrolysis is characterized very well, and allow for manufacturing has micro- for continuing antigen release within the extension time
Grain.Vaccine (including polypeptide, adjuvant etc.) can be encapsulated with delivery vector and be delivered.
In some applications, adjuvant or excipient may include in vaccine preparation.The selection of the adjuvant is logical by part
The method of application of vaccine is crossed to determine.The adjuvant used can also theoretically become known for appointing for the vaccine based on peptide or protein
What adjuvant.Adjuvant can be changed with the amount of assisting use, the amount with adjuvant, host animal and immunogene.
The immunogene of specific synthetic peptide combination epitope is provided in the following examples, for illustrating the present invention.
The purpose of these embodiments, which is only that, illustrates part of functions of the present invention, and any mode can not be constituted to the scope of the present invention
Limitation, application field of the invention are not limited to following embodiment.
As specific embodiment, the present invention provides a kind of resisting porcine circovirus synthetic peptides, pass through novel T1 and/or T2
Cell epitope sequence connects to form composition.Drug seeded with mammalian or birds are made with combination adjuvant in the composition, can make
Mammal or birds generate the antibody for pig circular ring virus whithin a period of time, pass through the pig circular ring virus antibody water of generation
Gentle specificity T fh quantity and frequency evaluate the ability of T1 and/or T2 epitope Help B Cells epitope.
Embodiment 1, the design of epitope combination
In combination epitope of the invention, T1 from Fusion protein of measles virus cd4 t cell epitope (Genebank:
AAF85664.1,a.a 292-304).This epitope is referred to as universal auxiliary type t cell epitope, can combine variety classes
Animal and the different MHC II molecule of allogenic animal, to activate the clone of more Tfh.
In combination epitope of the invention, B is SEQ ID NO:5 (embodiment 4, pig circular ring virus epitope capsid protein
(PCV) (GenBank:ACZ06596.1, capsid [Porcine circovirus-2], aa119-130 and aa231-233).
In combination epitope of the invention, cd4 t cell of the T2 from the hemagglutinin of influenza A virus (H3N8)
Epitope (GenBank:ABV58843.2, a.a 59-71)；This epitope is referred to as universal auxiliary type t cell epitope, can tie
Various animals and the different MHC II molecule of allogenic animal are closed, to activate the clone of a greater variety of Tfh.The present invention will
B cell epitope is placed between T1 and T2, it is therefore an objective to the B cell epitope that prevents the adjacent generation of T1 and T2 new and reduce target antibody
Generative capacity.
The present invention uses the heterogeneous general epitope of T1, T2 rather than identical general epitope, it is therefore an objective in conjunction with not of the same race
The MHC II of class, to activate wider Tfh precursor, i.e., infantilism () cd4 t cell.
In the present embodiment, the T1 sequence after modification is following SEQ ID NO:3 amino acid sequences: AKIKGVIVHRLAA
(SEQ ID NO:3).T1 sequence from Fusion protein of measles virus cd4 t cell epitope (Genebank:AAF85664.1,
A.a 292-304), sequence is SEIKGVIVHRLEG (SEQ ID NO:1).Newest discovery shows epitope peptide/MHC II
Compound and TCR affinity and Tfh differentiation be positively correlated.This affinity is embodied in two aspects, and 1) defined epitope peptide exists
Abundance on MHC II, this abundance depend on the affinity of epitope peptide and MHC II molecule；2) side chain and TCR of defined epitope peptide
Affinity.Based on this, inventor modifies SEIKGVIVHRLEG as follows, to improve the energy that it induces Tfh differentiation
Power:
(1) the 1st amino acid S is the anchor amino acids of MHC II, usually hydrophobic amino acid.In the present embodiment
In, by be replaced into the stronger A of hydrophobicity.
(2) deputy amino acid E is the binding site of TCR, and usually positively charged amino acid can promote the knot with TCR
It closes.In the present embodiment, by be replaced into positively charged amino acid K.
(3) amino acid of epitope C-terminal is MHC II anchor series, usually hydrophobic amino acid.In the present embodiment
In, E, G of 12,13 are replaced into the stronger A of hydrophobicity, to improve the anchoring ability of MHC II.
In the present embodiment, the T2 sequence after modification is following SEQ ID NO:4 amino acid sequences: AKFVKAWTLKLAA
(SEQ ID NO:4).Cd4 t cell epitope of the T2 sequence from the hemagglutinin of influenza A virus (H3N8)
(GenBank:ABV58843.2, a.a 59-71), amino acid sequence are PKYVKQNTLKLAT (SEQ ID NO:2).With T1
Modification it is similar, inventor modifies PKYVKQNTLKLAT as follows, with improve its induce Tfh differentiation ability:
(1) the 1st amino acid P is the anchor amino acids of MHC II, usually hydrophobic amino acid.In the present embodiment
In, by be replaced into the stronger A of hydrophobicity.
(2) the 3rd amino acid Y are the binding site of MHC II, are all that the F ratio Y of aromatic amino acid has stronger MHC
II binding ability.In the present embodiment, the 3rd Y is replaced into F.
(3) the 6th amino acid Q are the anchor amino acids of MHC II.In order to improve the anchoring ability of MHC II, by the 6th
The Q of position is replaced into the stronger A of hydrophobicity.
(4) the 7th amino acid Ns are the binding site of TCR, usually positively charged or aromatic amino acid can promote with
The combination of TCR.In the present embodiment, by be replaced into aromatic amino acid W.
(5) the 13rd amino acid T are the anchor amino acids of MHC II.In the present embodiment, 13 T are replaced into thin
Aqueous stronger A, to improve the anchoring ability of MHC II.
The synthesis of embodiment 2, polypeptide
With the peptide in chemical synthesis synthesis table 1.The present invention utilizes Merrifield solid phase synthesis process, uses ABI public affairs
The automatic Peptide synthesizer of 433A type is taken charge of, using WANG resin and 9-fluorenylmethyloxycarbonyl (Fmoc amido protecting group) and other protections
The amino acid of base group modification, according to from c-terminus (- COOH) to aminoterminal (- NH2) direction, successively synthesize synthetic peptide in table 1
Sequence.After the completion of synthesis, according to ABI company Peptide systhesis Standard cleavage program, polypeptide and resin is cut using hydrofluoric acid, is made
Polypeptide and resin separation, while also sloughing the blocking group of amino acid side chain.
The polypeptide being cleaved is quantified by washing and precipitating repeatedly by reversed-phase high performance liquid chromatography and mass spectrum
Analysis.After the peptide masses of analysis are qualified, weighing packing is subsequently used for downstream experiment.
The emulsification of embodiment 3, polypeptide
Serial No. SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ selected in precise table 1
ID NO:8 and SEQ ID NO:9 totally 5 polypeptides (referring to table 1) for being combined into peptide based immunogens, with dimethyl sub-maple (DMSO) and note
It penetrates and is dissolved with water, after ultrasound filtration, be configured to the polypeptide solution that concentration is 500 μ g/mL.Measure Total France of same volume
Company's white oil (pharmaceutical grade) is used as adjuvant, and the synthetic peptide based immunogens of Water-In-Oil are emulsified into Britain's IKA mulser.In addition one is prepared
Group does not contain the water for injection and Dao Daer white-oil adjuvant emulsification group of any antigenic substance, uses as negative control.Pass through matter
Amount is used for animal immune after examining.
Table 1, PCV virus epitopes and combinations thereof mode
Embodiment 4, pig circular ring virus epitope capsid protein (GenBank:ACZ06596.1, capsid [Porcine
Circovirus-2], aa119-130 and aa231-233)
1, immunization protocol
According to the good SEQ ID NO:5 of above-mentioned emulsification, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ
ID NO:9 amounts to 5 kinds of synthetic peptide based immunogens, 5 mouse is immunized using every kind of synthetic peptide based immunogens under the same conditions, under
Mouse is immunized in the test immunization protocol that face is listed:
Experimental group and size of animal: 6 groups, 5 groups of test group, 1 group of control group are divided into.
Experimental animal: C57 mouse, every group of 5 mouse.
Animal age, gender and weight: 6 week old male mices, 20 grams to 22 grams of weight.
Synthetic peptide based immunogens adjuvant and dosage form: Dao Daer white-oil adjuvant, Water-In-Oil.
Immunizing dose: every 0.2mL, every milliliter contains 500 μ g synthetic peptide based immunogens.
Blood sampling time: it is first 0th day immune, it takes a blood sample within the 7th day, the 15th day and the 30th day after being immunized.
Route of inoculation: double hind leg intramuscular injections, point 2 points of injections, every 0.1mL.
It is executed in strict accordance with above-mentioned mouse animal experiment scheme.ELISA (enzyme linked immunological is used in blood sampling separation serum storage
Adsorption test) experiment, determine Serum Antibody level.
2, indirect ELISA method measurement mice serum antibody level and result judgement
A. antibody determination.The flat enzyme mark in 96 holes is coated with preprepared sequence number SEQ ID NOs:5-9 synthetic peptide
Plate determines the antibody level that mouse generates by indirect ELISA method.It is dissolved with the 0.05M carbonate buffer solution of PH=9.6
Synthetic peptide SEQ ID NOs:5-9, concentration are 7 μ g/mL, and 100ul is added in every hole.4 degree are placed 16 hours.With 5% skimmed milk power
(sigma)/PBS closing, 37 degree keep the temperature 3 hours.It is analyzed at once for indirect ELISA after closing ELISA Plate.
Isolated mice serum is first done into 50 times of dilutions, is then serially diluted until the 8th arranges hole, first row i.e. 50 times for 3 times
Dilution, serum dilution are shown in Table 2.100 μ l of sample is added in every hole, concurrently sets negative control.It is incubated for 1 hour.After washing, enzyme is added
Secondary antibody is marked, every 100 μ l of hole is incubated for 1 hour.Tmb substrate is added after washing to react 15 minutes, terminate liquid is added.In wavelength 450nm
Measure the OD value in each hole of ELISA Plate.
Table 2, serum samples diluted multiple table
Note: X1 to X96 is the sample OD value specifically measured.
Result judgement standard: the wavelength 450nm cutoff value absorbed is set as by the linear regression analysis based on trap
0.5, which is correct and accurate, because the OD value for carrying out analysis dilution normal control mice sample every time is respectively less than
0.15.We measure the extension rate of each group mouse blood serum sample in OD value with Reed-Muench method.
Reed-Muench method formula:
Sample to be tested=beforehand dilution multiple × 3k
K=n+ (X1-0.5)/(X1-X2)
X1 is higher than the OD value of the sample maximum dilution multiple of cutoff value 0.5
X2 is lower than the sample extension rate OD value of cutoff value 0.5
Weighted average is calculated later, and final result is shown in Table 3.Illustrate immune effect if serum samples diluted degree is higher
Better.
Antibody test result after mouse, extension rate table is immunized in 5 kinds of table 3, PCV vaccine peptide based immunogens
A indicates lower than 50 times dilutions, and cutoff value 0.5 is less than in this dilution range, negative.Therefore it is not processed.
B. result judgement.The group of the mean OD value for measuring every group of mouse through indirect ELISA visible T1-B-T2 and T2-B-T1
It closes object and is significantly better than other each groups.
From figure 1 it appears that the result of measurement can intuitively reflect the height of antibody level, it is auxiliary can also to embody T
The effect of synergidae epitope is strong and weak.Sequence SEQ ID NO:5 does not generate antibody, shows if antigen does not have t helper cell epitope
It would not cause antibody response；The antibody dilution times that SEQ ID NO:8 and SEQ ID two synthetic peptide combinations of NO:9 generate
6 to 8 times of number more than SEQ ID NO:6 and SEQ ID NO:7 composition.T cell epitope is directly related to the generation of antibody, good
Good t cell epitope design can generate high antibody response.
By interpretation of result, it will be seen from figure 1 that synthetic peptide SEQ ID NO:5 epitope group, combined peptide T1-B group SEQ ID
NO:6 and B-T2 group SEQ ID NO:7 mean OD value is well below T1-B-T2 group SEQ ID NO:8 and T2-B-T1SEQ ID
The mean OD value of NO:9 illustrates that both combined peptides of T1-B-T2 and T2-B-T1 have best immune effect.
3, using the quantity and frequency of flow cytometry evaluation Tfh cell
The sterile mouse spleen adopted after immune 8 days, is added the erythrocyte cracked liquid of 2mL, is squeezed out with 5mL syringe tail portion
Spleen cell is added 2% serum RPMI-1640 culture solution of 5mL and is resuspended, and 1800 turns are centrifuged 6 minutes, abandons supernatant.Cell precipitation is used
2% serum RPMI-1640 culture solution is resuspended.After cell count, with the various anti-Tfh cell marker molecules from BD company
Antibody (CD4, CD44, CXCR5) marks cell, operates dyeing according to the BD application program of standard, then uses BD flow cytometer
Canto II measurement result.
Result judgement standard: in the Tfh cell of every mouse of measurement frequency and every spleen shared in cd4 t cell
Tfh quantity.
(1) frequency of different cd4 t cells and B cell epitope combination induction Tfh: contain different cd4 t cells and B cell epitope
Mouse is immunized after 8 days in combined synthetic peptide, takes spleen, removes red blood cell, the full cell of separating spleen, using for CD4, CD44,
The fluorescent labeled antibody of CXCR5 is dyed.Tfh cell is defined as the cell mass of CD4+CD44+CXCR5+.Rectangle in Fig. 2
The numerical value of top is shown as the frequency of Tfh cell.The frequency the high, illustrates the immune effect of synthetic peptide more preferably.It can from Fig. 2
Out, the frequency of T1-B-T2 group SEQ ID NO:8 and T2-B-T1SEQ ID NO:9 is significantly larger than synthetic peptide SEQ ID NO:5 table
The frequency of hyte, combined peptide T1-B group SEQ ID NO:6 and B-T2 group SEQ ID NO:7, illustrate T1-B-T2 and T2-B-T1 this
Two kinds of combined peptides have best immune effect.
(2) quantity of different cd4 t cells and B cell epitope combination induction Tfh: complete according to the frequency of cell and spleen
The quantity of cell, calculates the quantity of Tfh in each mouse spleen as follows: Tfh cell quantity=splenocyte quantity ×
Cd4 t cell frequency × CD44+CXCR5+ cell frequencies.The quantity the more, illustrate the immune effect of synthetic peptide more preferably.It can from Fig. 3
To find out, sequence SEQ ID NO:5 does not generate antibody, shows if antigen does not have t helper cell epitope that would not cause antibody
Response.T cell epitope is directly related to the generation of antibody, and good t cell epitope design can generate high antibody response.T1-
The frequency of B-T2 group SEQ ID NO:8 and T2-B-T1SEQ ID NO:9 is significantly larger than synthetic peptide SEQ ID NO:5 epitope group, group
The quantity for closing peptide T1-B group SEQ ID NO:6 and B-T2 group SEQ ID NO:7 illustrates both combinations of T1-B-T2 and T2-B-T1
Peptide has best immune effect.
Those skilled in the art is it should be understood that although for illustrative purposes, this document describes tools of the invention
Body embodiment, but it can be carry out various modifications without departing from the spirit and scope of the present invention.Therefore, of the invention specific
Embodiments and examples should not be considered as limiting the scope of the invention.The present invention is limited only by the appended claims.This Shen
Please in quote all documents be fully incorporated herein by reference.
Claims (13)
1. a kind of polypeptide is made of the amino acid sequence that T1-B-T2 or T2-B-T1 configuration arranges,
Wherein, T1 is amino acid sequence shown in SEQ ID NO:3；
B is SEQ ID NO:5；
T2 is amino acid sequence shown in SEQ ID NO:4.
2. polypeptide as described in claim 1, wherein the amino acid sequence of T1-B-T2 or T2-B-T1 configuration arrangement is
SEQ ID NO:8 or SEQ ID NO:9.
3. polypeptide as claimed in claim 1 or 2, by it is artificial synthesized, base is expressed or passed through by protokaryon or eukaryotic
It is obtained because recombinant technique is embedded in pathogen.
4. a kind of isolated polynucleotides, it includes one of be selected from the group:
A) the multicore sweet acid of polypeptide as described in claim 1 is encoded；Or
B) polynucleotides complementary with polynucleotides a).
5. a kind of vaccine, it includes at least one polypeptides as claimed in claim 1 or 2.
6. vaccine as claimed in claim 5 also includes medicine or veterinarily acceptable delivery vector or adjuvant.
7. such as vaccine described in claim 5 or 6, administration method be selected from intraocular, intranasal, intramuscular, injection, it is oral, intraperitoneal,
Subcutaneously, local, intradermal and transdermal delivery.
8. a kind of vaccine adjuvant, it includes at least one polypeptides as claimed in claim 1 or 2.
9. polypeptide as claimed in claim 1 or 2 is in preparation in the preventative vaccine of resisting porcine circovirus, therapeutic vaccine
Purposes.
10. polypeptide as claimed in claim 1 or 2 is preparing the purposes in monoclonal or polyclonal antibody.
11. polynucleotides as claimed in claim 4 are preparing the purposes in polypeptide.
12. the preventative vaccine, therapeutic such as the described in any item vaccines of claim 5-7 in preparation for resisting porcine circovirus
Purposes in vaccine.
13. preventative vaccine, therapeutic vaccine that vaccine adjuvant as claimed in claim 8 is used for resisting porcine circovirus in preparation
In purposes.
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| EP2143447A1 (en) * | 2008-07-11 | 2010-01-13 | Universität Konstanz | Bioconjugates comprising Aß-autoantibody-specific epitopes for active immunotherapy and diagnosis of Alzheimer's disease |
| CN103108653A (en) * | 2010-07-08 | 2013-05-15 | 美国联合生物医学公司 | Designer peptide-based pcv2 vaccine |
| CN103536912A (en) * | 2013-09-30 | 2014-01-29 | 重庆理工大学 | Porcine circovirus II-type (PCV2) epitope peptide vaccine and preparation method thereof |
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| EP2143447A1 (en) * | 2008-07-11 | 2010-01-13 | Universität Konstanz | Bioconjugates comprising Aß-autoantibody-specific epitopes for active immunotherapy and diagnosis of Alzheimer's disease |
| CN103108653A (en) * | 2010-07-08 | 2013-05-15 | 美国联合生物医学公司 | Designer peptide-based pcv2 vaccine |
| CN103536912A (en) * | 2013-09-30 | 2014-01-29 | 重庆理工大学 | Porcine circovirus II-type (PCV2) epitope peptide vaccine and preparation method thereof |
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| Plasmodium falciparum synthetic LbL microparticle vaccine elicits protective neutralizing antibody and parasite-specific cellular immune responses.;Thomas J. Powell et al.;《Vaccine》;20131231;1898-1904 |
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