CN105175550A - Synthetic peptide vaccine for resisting porcine circovirus and application of synthetic peptide - Google Patents
Synthetic peptide vaccine for resisting porcine circovirus and application of synthetic peptide Download PDFInfo
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Abstract
The invention provides a polypeptide. The polypeptide comprises amino acid sequences adopting T1-B-T2 or T2-B-T1 configuration arrangement, wherein T1 is modified SEQ ID NO: 1; B is SEQ ID NO: 5; T2 is modified SEQ ID NO: 2. The invention further provides an application of the polypeptide in preparation of preventive vaccines and therapeutic vaccines for resisting a porcine circovirus. The polypeptide can be used for synthetic peptide vaccines or other types of vaccines and can improve the immunogenicity of the vaccine.
Description
Technical field
The present invention relates to field of biological product, particularly a kind of synthetic peptide vaccine for resisting porcine circovirus and uses thereof.
Background technology
Antibody has important effect in anti-infectious immunity.Antibody directly and pathogenic agent combine, prevention pathogenic infection normal cell; Antibody can also and bacteriotoxin effect, block detoxifying function in target cell.In addition, antibody also can be adsorbed in pathogenic agent surface, thus the approach of activating complement mediation carrys out cracking pathogenic agent or promotes phagocytic cell, as scavenger cell, addicted to the granulocytic phagolysis in center and the cell-mediated ADCC effect of natural killer.In addition, antibody, particularly IgG, can also enter the recycle system of fetus through placenta materna, thus the infection of protection fetus at the birth initial stage from pathogenic agent.Because the anti-infectious function that antibody is powerful, be understood that the vaccine for man Vaccine effectiveness of nearly all listing all relevant to antibody horizontal (Vaccines, 6thedition, S.A.Plotkin).
Antibody can disease-resistant former invasion and attack thus shield to body, how to improve the immunogenicity of vaccine thus the response level strengthening antibody becomes the critical problem of vaccine research and development field.The secretory cell of antibody is called plasmocyte, and it derives from the B cell of activation, is particularly positioned at the B cell (GerminalCenterBcells, GCB) of Lymphoid tissue germinal center.GCB cytodifferentiation plasmablast needs the lasting stimulation of antigen.Adjuvant component in inactivated vaccine or subunit vaccine is largely by slowly-releasing antigen, thus provides lasting antigenic stimulation to GCB cell, promotes that it is divided into the plasmocyte of secretory antibody.Except antigenic stimulation, GCB cell also needs " help " of cd4 t cell that activate, homology (cognate) just can be divided into plasmocyte.The cd4 t cell activated is divided into the function subclass different with phenotype under different immune microenvironments, comprises TH1, TH2 and TH17 etc., and these cd4 t cells activated are commonly called helper T cell (helperTcell, Th).Wherein TH1 and TH2 has " help " B cell and is divided into plasmacytic ability.
Up-to-date research finds, specialty provides the helper T cell of B cell " help " to be a novel subclass being different from TH1 and TH2.Because this subclass expresses Chemokine Receptors CXCR5 at cell surface height, after the recruitment by Chemokines CC XCL13, migrate to B cell follicular regions and be called as folliculus helper T cell (FollicularhelperTcell).In folliculus district, the Tfh cell of activation provides CD40L, ICOS, IL-21 etc. " help " signal to GCB cell, guarantees the survival of GCB cell and promotes that it is divided into plasmocyte.And B cell is for obtaining " help " of Tfh cell, must discernible for the TCR of Tfh epitope peptide section be placed on MHCII, and be illustrated in cell surface for TCR combination to activate Tfh cell.To sum up, B cell is divided into the plasmocyte of secretory antibody, successively need three steps: 1) B cell utilizes B-cell receptor (Bcellreceptor, BCR) specifically identify, conjugated antigen, thus activate BCR signal path, B cell is made to be in active state, for cell proliferation subsequently and plasma cell differentiation are prepared; 2) after antigen is combined with BCR, with the mode of endocytosis (Endocytosis) be swallowed to B cell formed in bite body (Endosomes), interior bite the low pH condition of body under, antigen is degraded, block into 13-25 amino acid whose small peptide, these small peptides are placed on the MHCII molecule of B cell, and being transported to cell surface, the TCR for Tfh identifies.3) after the TCR of Tfh identifies the antigen of B cell mhc class ii molecule submission, be combined with the B7 molecule of B cell by the costimulatory molecule CD28 on film and activate, the Tfh cell upregulation of activation expresses CD40L, this molecule is combined with B cell membrane receptor CD40 stimulates B cell further, and B cell is activated completely.So induce and at least comprise two parts compared with the antigen of High antibody level, one is the antigenic component that can identify for B cell BCR, and be then the t cell epitope that can be identified by TCR in addition, Neither of the two can be dispensed.
All karyocyte surfaces all distribute mhc class i molecule, and mhc class ii molecule is mainly distributed in professional antigen presenting cell film, as B cell, dendritic cell, scavenger cell etc.Mhc class ii molecule be combined into mixture (pMHC) with certain avidity and antigen peptide and submission in cell surface, Tfh cell utilizes the specific identification of TCR, is activated in conjunction with this mixture.Threaten the Pathogen category of the mankind various, and often make a variation, thus the height polymorphism of mhc gene and polygene have greatly expanded the individual scope to antigen peptide submission, improve the individual defensive ability/resistance ability to adverse environment, are conducive to maintaining population existence and continuity.Current research finds, the avidity of antigen peptide and mhc class ii molecule is higher, and both incorporation ranges are wider, TCR identification, stronger in conjunction with the ability of pMHC, then generation responsiveness Tfh cell mass is more, and the immune efficacy of generation is stronger.Therefore, develop a kind of t cell epitope antigen peptide mhc class ii molecule to general avidity, become the key subjects of current vaccines development.
Early stage experiment is verified, and the generation of antibody needs " help " of cd4 t cell.Based on this, usually the haptens composition covalent coupling of B cell epi-position or other chemical small molecule class to carrier proteins, as on KLH, BSA, OVA, these carrier proteinss BCR identify and conjugated antigen after, with target antigen by endocytosis in B cell, thus processed, be showed on MHCII, then submission is to cd4 t cell, cd4 t cell is activated, and corresponding B cell, after acquisition activation cd4 t cell helps, is divided into they produce antibodies.In addition, in vaccinology field, usually using the composition of B cell epi-position and cd4 t cell epi-position as antigen inoculation body, make B cell produce antibody under the help of cd4 t cell, thus play the effect of immunoprotection.Although the animal aftosa synthetic peptide vaccine comprising cd4 t cell epi-position and B cell epi-position goes on the market, this type of synthetic peptide vaccine major part is also under test, and immune effect need discussion.Up to now, people is not yet had to go on the market with synthetic peptide vaccine, an important reason is scarcely out of swaddling-clothes to the understanding of Tfh cell and B cell mutual relationship, and designed synthetic peptide vaccine stimulates the quality and quantity of the cd4 t cell (i.e. Tfh) of the B cell that can help to have much room for improvement.
Summary of the invention
An aspect of of the present present invention provides a peptide species, and it comprises the aminoacid sequence of T1-B-T2 or T2-B-T1 configuration arrangement,
Wherein, T1 is the SEQIDNO:1 after modifying;
B is SEQIDNO:5;
T2 is the SEQIDNO:2 after modifying.
T1, B or T2 can carry out functional connection by intervening sequence, and wherein intervening sequence can be any aminoacid sequence not affecting T1, B or T2 structure or function.In one embodiment of the invention, intervening sequence is ε K.
In an embodiment of the present invention, described modification comprises displacement, adds and/or lacks the one or more amino acid in original cell epitope, and the cell epitope after modifying has identical immunologic function with described original cell epitope.
Represent in embodiment of the present invention, the modification of described SEQIDNO:1 can comprise following one or more modifications:
(1) the 1st amino acids Serine is hydrophobic amino acid;
(2) the 2nd amino acids L-glutamic acid are replaced into the amino acid of positively charged;
(3) the 12nd amino acids L-glutamic acid are replaced into hydrophobic amino acid; With
(4) the 13rd amino acids glycine are replaced into hydrophobic amino acid.
Represent in embodiment of the present invention, the modification of described SEQIDNO:2 comprises following one or more modifications:
(1) the 1st amino acids proline(Pro) is replaced into hydrophobic amino acid;
(2) the 3rd amino acids tyrosine substitution are other die aromatischen Aminosaeurens;
(3) the 6th amino acids glutamine are replaced into hydrophobic amino acid;
(4) the 7th amino acids l-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 epi-position comprises the aminoacid sequence shown in SEQIDNO:3.
In one particular embodiment of the present invention, T2 epi-position comprises the aminoacid sequence shown in SEQIDNO:4.
Wherein, above-mentioned epi-position is combined through synthetic, by protokaryon or eukaryotic cell expression or embed pathogenic agent by gene recombination technology and obtain.
In another aspect of the present invention, provide a kind of polynucleotide of separation, it comprises the one be selected from lower group:
A) to encode polypeptide as elucidated before; Or
B) with the polynucleotide of polynucleotide a) complementary or stingent hybridization.
In another aspect of the present invention, provide a kind of vaccine, it comprises the foregoing polypeptide of at least one.
In the present invention, vaccine of the present invention also comprises medical science or veterinarily acceptable delivery vector or adjuvant.
In an embodiment of the present invention, route of vaccine administration be selected from intraocular, in nose, intramuscular, injection, oral, intraperitoneal, subcutaneous, locally, intracutaneous and transdermal delivery.
In one aspect of the invention, provide a kind of vaccine adjuvant, it comprises the foregoing polypeptide of at least one.
In addition, polypeptide of the present invention is for the preparation of the purposes in the preventative vaccine of resisting porcine circovirus, therapeutic vaccine.And isolated polypeptide according to the present invention is also in category of the present invention for the production of the purposes of polynucleotide.
In the present invention, polypeptide of the present invention is preparing the purposes in mono-clonal or polyclonal antibody.
In the present invention, polynucleotide of the present invention are preparing the purposes in polypeptide.
In the present invention, vaccine of the present invention is for the preparation of the purposes in the preventative vaccine of resisting porcine circovirus, therapeutic vaccine.
In the present invention, vaccine adjuvant of the present invention is for the preparation of the purposes in the preventative vaccine of resisting porcine circovirus, therapeutic vaccine.
Beneficial effect of the present invention
Pass through embodiment, contriver finds that T1-B-T2 induces T1-B or T2-B or B height 5-10 times that the energy force rate of body generation antibody is single, this finds to illustrate that combined sequence of the present invention can stimulate body to produce stronger Tfh response, the more important thing is, the responsibility strengthening Tfh can promote that corresponding antibody produces significantly.Of the present inventionly comprise preventative vaccine, the therapeutic vaccine that epi-position combination product can be used in anti-pathogen infection or tumour or autoimmune disorder widely, produce high-caliber antibody to induce; This combined sequence also can be used for producing polyclone or monoclonal antibody, for diagnosis, treatment and scientific research.Because the present invention is artificial synthetic product, there is definite ingredients, quality controllable, target clearly feature, for the synthesis of peptide vaccine, there is stronger advantage.
Below in conjunction with accompanying drawing, the present invention will be described in more detail.From detailed description hereafter, above-mentioned aspect of the present invention and other aspects of the present invention will be obvious.
Accompanying drawing explanation
Fig. 1 illustrates the result of serum diluting multiple after PCV vaccine immune mouse.
Fig. 2 illustrates the frequency of different cd4 t cell and B cell epi-position combination induction Tfh.
Fig. 3 illustrates the quantity of different cd4 t cell and B cell epi-position combination induction Tfh.
Embodiment
Unless stated otherwise, term of the present invention has the normally used implication in this area.
Epi-position can be categorized as B cell type, T cell type or B cell type and T cell type, depends on the type of the immunne response that they cause.The definition of B cell or t cell epitope is not clear and definite; Such as, a kind of peptide epitopes can produce by induction of antibodies, but this epi-position can have a kind of sequence simultaneously, and this sequence can be incorporated into people HLA molecule, makes it easily arrive CTL, be therefore that two B cell and T cell are classified for this defined epitope." t helper cell " or " Tfh " is any T cell that release promotes B cell and the activation of killer T cell and the cytokine of function when being stimulated by specific antigens.
" amino acid " refers to compound, and it has amino group and hydroxy-acid group, and 1, the 2-replacement, 1,3-preferably on carbon skeleton replaces or Isosorbide-5-Nitrae-substitute mode.A-amino acid is most preferred, and (they are L-amino acid to be included in the 20 kinds of natural amino acids found in albumen, except glycine), corresponding D-amino acid, corresponding N-methylamino acid, the amino acid that side chain is modified, amino acid (the such as 4-hydroxy-proline that the biosynthesizing do not found in albumen obtains, 5-hydroxyl-Methionin, citrulline, ornithine, canavanine, djenkolic acid, beta-cyano L-Ala), and a-amino acid (the such as aminoisobutyric acid that synthesis is derivative, nor-leucine, norvaline, homocysteine and homoserine).L-Ala and y-aminobutyric acid are 1,3-amino acid and Isosorbide-5-Nitrae-amino acid whose example respectively, and other amino acid much is well known in the art.(comprise two amino acid whose dipeptides, wherein CONH key is by CHOHCH for the structure things such as the structure things such as statine sample (comprise two amino acid whose dipeptides, wherein CONH key is replaced by CHOH), hydroxy ethylene
2displacement), (comprise two amino acid whose dipeptides, wherein CONH key is by CH for the structure thing such as acid amides of reduction
2nH key is replaced) and the structure thing (comprise two amino acid whose dipeptides, wherein CONH key is replaced by CSNH key) such as thioamides be also the useful amino-acid residue of the present invention.For amino acid of the present invention be can business obtain or by conventional synthesis process obtain amino acid.
" displacement " refers to by different amino acid or the one or more amino acid of nucleotide subsitution or Nucleotide.
" disappearance " refers to the disappearance of one or more amino acid or Nucleotide in aminoacid sequence or nucleotide sequence.
" insertion " or " interpolation " refers to and causes compared with natural existence or the molecule before changing in the change in aminoacid sequence or nucleotide sequence, the increase of one or more amino acid or Nucleotide.
" replace, lack and/or add one or more amino acid " then to refer to, the amino acid of the number of the degree utilizing the displacement of the manufacture method of the known mutant nucleic acids such as kunkel method Kunkel or polypeptide, disappearance and/or interpolation can replace, lack and/or add or Nucleotide.Said mutation is not limited to the sudden change utilizing known fixed-point mutation method and human-induced, also can carry out separation and purification to the sudden change of naturally occurring nucleic acid or protein and obtain.To amino acid whose sudden change, can to include one or more amino-acid residue be conformation is the amino acid of D-type, and the rare amino acid that nature exists or manually modified amino acid, these amino acid can may not be is encoded by genetic codon.Similar therewith, induction nucleic acid is undergone mutation, and can comprise the naturally occurring Nucleotide of nature, also can comprise the Nucleotide with modification.
Amino acid whose conservative substituting well known by persons skilled in the art is within the scope of the invention.Conserved amino acid substitutes and comprises an amino acid and have identical types of functional groups or side chain such as aliphatics, aromatics, positively charged, electronegative amino-acid substitution with another.These substitute can strengthen oral administration biaavailability, penetrates into central nervous system, target specific cells colony etc.Those skilled in the art know, in encoding sequence, change, increase or delete single amino acids or little percentile amino acid whose to substitute peptide, polypeptide or protein sequence indivedual, delete or increase be " conservative modify variant ", wherein change and result in amino acid replacement like amino acid chemical classes.Such as, below six groups separately containing for the conservative amino acid substituted each other: 1) L-Ala (A), Serine (S), Threonine (T); 2) aspartic acid (D), L-glutamic acid (E); 3) l-asparagine (N), glutamine (Q); 4) arginine (R), Methionin (K); 5) Isoleucine (I), leucine (L), methionine(Met) (M), α-amino-isovaleric acid (V); And 6) phenylalanine (F), tyrosine (Y), tryptophane (W).There is provided functionally similar amino acid whose conservative alternative to be known in the art, other any known alternative are all fine.
" hydrophobic amino acid " refers to that side chain has the amino acid of high hydrophobicity, such as methionine(Met) (M), tryptophane (W), tyrosine (Y), phenylalanine (F), α-amino-isovaleric acid (V), leucine (L), Isoleucine (I), proline(Pro) (P) and L-Ala (A).
" die aromatischen Aminosaeuren " refers to the amino acid containing aromatic nucleus, such as tyrosine (Y), phenylalanine (F), tryptophane (W) and thyroxine.
" amino acid of positively charged " refers to the amino acid of side chain with positive charge, such as Methionin (K), arginine (R) and Histidine (H).
" polypeptide " or " aminoacid sequence " refers to peptide, oligopeptides, polypeptide or protein and Partial Fragment thereof, therebetween with the amino acid that peptide bond is connected.When " aminoacid sequence " in the present invention relates to a kind of aminoacid sequence of naturally occurring protein molecule, this " polypeptide " or " protein " does not also mean that aminoacid sequence is restricted to the complete natural acid sequence relevant to described protein molecule.Aminoacid sequence of the present invention can containing additional peptide.As additional peptide, the peptide as Epitope tags such as polyhistidine tag (His-tag) or Myc, FLAG is example.
Peptide analogs and peptide mimics are also included within scope of the present invention, comprise salt and the ester of peptide of the present invention in addition.According at least one blocking groups that peptide analogs of the present invention optionally comprises at least one alpha-non-natural amino acid and/or holds at C end or N.The salt of peptide of the present invention is acceptable organic salt and inorganic salt on physiology.The design of suitable " analogue " can use area of computer aided.
" peptide mimics " refers to that peptide according to the present invention is modified to comprise this mode of at least one non-peptide bond as urea key, amino-formate bond, sulphonamide key, hydrazine key or other covalent linkage any.The design of suitable " peptide mimics " can be assisted with computer.
Salt and the ester of peptide of the present invention comprise within the scope of the invention.The salt of peptide of the present invention is acceptable organic salt and inorganic salt on physiology.The functional deriv of peptide of the present invention comprises the derivative can prepared by the functional group of rolling into a ball existence as the side chain on residue or N or C end group by manner known in the art, and comprise in the present invention, as long as their keep pharmaceutically acceptable, namely they do not destroy the activity of peptide and do not give containing its composition with toxicity.These derivatives such as comprise carboxyl aliphatic ester, by with ammonia or primary amine or secondary amine react the carboxyl produced acid amides, by with acyl moiety (such as alkyloyl or carbocyclic aroyl) react the free amine group of the amino-acid residue formed N-acyl derivative or by reacting the O-acyl derivative of the free hydroxyl group (such as seryl or threonyl residues) formed with acyl moiety.
" polynucleotide ", " nucleotide sequence " or " base sequence " refer to Nucleotide, oligonucleotide or polynucleotide and fragment thereof or part.Nucleic acid of the present invention can exist with the form (such as, cDNA or genomic dna) of the form of RNA (such as, mRNA) or DNA.DNA can be double-strand, also can be strand.Single stranded DNA or RNA can be any one in coding strand (sense strand) or noncoding strand (antisense strand).In addition, polynucleotide of the present invention also can merge the polynucleotide of code tag mark (sequence label or marker sequence) at its 5 ' end or 3 ' end.They can be synthesis or from natural origin obtain (such as, be separated and/or purifying), it can comprise Nucleotide that is natural, non-natural or modified, and it can comprise the key between Nucleotide that is natural, non-natural or that change, such as phosphoramidic acid ester bond or phosphorothioate bond, be used for replacing the phosphodiester bond that exists between the oligonucleotide nucleotide of unmodified.
" separation " one word refer to material separated from its original environment (such as, if spontaneous just refer to its natural surroundings).Such as it is exactly be not separated that spontaneous polynucleotide or polypeptide are present in Live Animals, and same polynucleotide or polypeptide are separately separated with some or all materials coexisted with it in natural system.Such polynucleotide or polypeptide can be parts for a certain carrier, also can be parts for a certain composition.Since carrier and composition are not the compositions of its natural surroundings, they remain separation.
" nucleic acid hybridization " is well known in the art (see, such as, Sambrook etc., MolecularCloning:ALaboratoryManual, 3rdEd., ColdSpringHarborLaboratory, 2001).Usually, temperature is higher, and salt concn is lower, then preciseness becomes higher (being difficult to hybridization), thus can obtain more identical polynucleotide.The hybridization temperature be applicable to is different according to the length of base sequence or its base sequence.In addition, the invention still further relates to and hybridize under " stringent condition ".In the present invention, " stringent condition " refers to, compared with the hybridization under low ionic strength and comparatively high temps and wash-out, such as, overnight incubation under the condition of 42 DEG C, in (denatured sheared salmon sperm dnas of the sodium phosphate (pH7.6) of 50% methane amide, 5 × SSC (150mMNaCl, 15mM trisodium citrate), 50mM, 5 × Denhardt solution, 10% T 500 and 20 μ g/mL), then under 65 DEG C of conditions with 0.1 × SSC wash-out.
Vaccine of the present invention comprises polypeptide or comprises the recombination fusion protein of polypeptide and optional adjuvant.Described vaccine can be formulated for and use one of in a multitude of different ways.According to one embodiment of the invention, described vaccine intranasal administration.Described vaccine preparation can be applied to Lymphoid tissue in any convenient manner.But, preferably it is applied on the wall of nasal passage as liquid stream or drop.Intranasal compositions such as can be formulated as nasal drop, sprays in liquid form, or is suitable for sucking, and is formulated as pulvis, creme or emulsion.Said composition can contain multiple additives, such as adjuvant, vehicle, stablizer, buffer reagent or sanitas.
In order to simple application, described vaccine is preferably being suitable for supplying in the container with polypeptide described in nasal drop or aerocolloidal formal distribution or recombination fusion protein.In some preferred embodiment, described vaccine is formulated for mucosal delivery, especially nasal delivery (Arnon etc., Biologicals (biological products) .2001; 29 (3-4): 237-42; Ben-Yedidia etc., IntImmunol. (Interaational) 1999; 11 (7): 1043-51).
In one embodiment of the invention, undertaken using through eye by spraying, aerosol or eye drops.Can be liquid or dry powder by vaccine formulation, to use as aerosol, sprays or eye drops.The composition used as aerosol, eye drops or sprays can comprise the vehicle be generally comprised within such composition of one or more types, such as sanitas, viscosity modifier, tension regulator, tear blocker, buffer reagent, stablizer, carrier, adjuvant etc., to prepare the preparation used through eye.
In one embodiment of the invention, use as oral, and described vaccine such as can provide with the form of tablet, or wrap up in gelatine capsule or microcapsule.Also in still another embodiment, described vaccine is formulated for parenteral and uses.In some embodiments, described vaccine is formulated for large-scale inoculation, such as, use together with jet injector or disposable medication tube.Also according to another embodiment, described in use be intramuscular.Also according to another embodiment, described in use be intracutaneous.The pin that specialized designs is used for intracutaneous input vaccine is well known in the art, such as, be particularly disclosed in U.S. Patent number 6,843,781 and 7,250, in 036.According to other embodiment, described in use and carry out with needleless injector.
The preparation of these forms is general knowledge to those skilled in the art.
Delivery vector comprises liposome, microparticle, nano particle etc.Liposome provides for antigen delivery and another delivery system presented.The bilayer vesicle that liposome is made up of the phosphatide at the usual moisture center of encirclement one and other sterol, described moisture in antigen or other product can be encapsulated in the heart.Liposome structure is highly various, and many types are from about 25nm to the nano level of about 500 μm in micron-sized scope.Have been found that liposome can effectively delivering therapeutic agents to skin and mucomembranous surface.Liposome can be modified further, for such as carrying out targeted delivery by specific antibody is incorporated into surface film, or is changed to encapsulating bacterium, virus or parasite.The mean survival time of complete liposome structure or transformation period can extend when comprising some polymkeric substance such as polyoxyethylene glycol, allow to extend release in vivo.Liposome can be individual layer or multilayer.Microparticle and nano particle use little biodegradable spheroid, and it is used as the reservoir of vaccine delivery.The major advantage of what polymer microballoon had the exceed adjuvant of other Reservoir effect is, they are extremely safes, and are used for human medical by U.S. food and drugs administration approved as the suture line be applicable to and are used as biodegradable drug delivery system.The speed of multipolymer hydrolysis is characterized very well, and then allows to be manufactured in time expand the microparticle with the release of lasting antigen.Vaccine (comprising polypeptide, adjuvant etc.) can be sent with delivery vector encapsulating.
In some applications, adjuvant or vehicle can be included in vaccine preparation.The method of application of part by vaccine decides by the selection of described adjuvant.The adjuvant used can also be any adjuvant of the vaccine become known for based on peptide or albumen in theory.Adjuvant uses with the amount of assisting a ruler in governing a country, and this amount can change with adjuvant, host animal and immunogen.
The immunogen of concrete synthetic peptide combination epi-position is provided, for illustrating the present invention in the following examples.The object of these embodiments is only part of functions of the present invention is described, can not form the restriction of any mode to scope of the present invention, Application Areas of the present invention is not limited to following embodiment.
As specific embodiment, the invention provides a kind of resisting porcine circovirus synthetic peptide, it forms composition by novel T1 and/or T2 cell epitope sequence series connection.Said composition coordinates adjuvant to make medicine seeded with mammalian or birds, can make Mammals or birds within for some time, produce antibody for pig circular ring virus, by the pig circular ring virus antibody horizontal that produces and specificity T fh quantity and frequency, evaluate the ability of T1 and/or T2 epi-position Help B Cells epi-position.
The design of embodiment 1, epi-position combination
In combination epi-position of the present invention, T1 derives from the cd4 t cell epi-position (Genebank:AAF85664.1, a.a292-304) of Fusion protein of measles virus.This epi-position is called as universal auxiliary type t cell epitope, and it in conjunction with various animals and the different MHCII molecule of allogenic animal, thus can activate the clone of more Tfh.
In combination epi-position of the present invention, B is SEQIDNO:5 (embodiment 4, pig circular ring virus epi-position capsid protein (PCV) (GenBank:ACZ06596.1, capsid [Porcinecircovirus-2], aa119-130 and aa231-233).
In combination epi-position of the present invention, T2 derives from the cd4 t cell epi-position (GenBank:ABV58843.2, a.a59-71) of the hemagglutinin of influenza A virus (H3N8); This epi-position is called as universal auxiliary type t cell epitope, and it in conjunction with various animals and the different MHCII molecule of allogenic animal, thus can activate the clone of a greater variety of Tfh.B cell epi-position is placed between T1 and T2 by the present invention, and object is the generative capacity preventing the new B cell epi-position of the adjacent generation of T1 and T2 and reduce target antibody.
The present invention adopts the heterogeneous general epi-position of T1, T2 instead of identical general epi-position, and object is in conjunction with different types of MHCII, thus activates Tfh precursor widely, namely juvenile form (
) cd4 t cell.
In the present embodiment, the T1 sequence after modification is following SEQIDNO:3 aminoacid sequence: AKIKGVIVHRLAA (SEQIDNO:3).T1 sequence derives from the cd4 t cell epi-position (Genebank:AAF85664.1, a.a292-304) of Fusion protein of measles virus, and its sequence is SEIKGVIVHRLEG (SEQIDNO:1).Up-to-date discovery shows, the differentiation positive correlation of the mixture of epitope peptide/MHCII and the avidity of TCR and Tfh.This avidity is embodied in two aspects, and 1) abundance of defined epitope peptide on MHCII, this abundance depends on the avidity of epitope peptide and MHCII molecule; 2) side chain of defined epitope peptide and the avidity of TCR.Based on this, inventor modifies as follows to SEIKGVIVHRLEG, to improve the ability of its induction Tfh differentiation:
The amino acid S of (1) the 1st is the anchor amino acids of MHCII, is generally hydrophobic amino acid.In the present embodiment, it is by for being replaced into the stronger A of hydrophobicity.
(2) deputy amino acid E is the binding site of TCR, and the amino acid of usual positively charged can promote the combination with TCR.In the present embodiment, it is by the amino acid K for being replaced into positively charged.
(3) amino acid of epi-position C-terminal is MHCII anchor series, is generally hydrophobic amino acid.In the present embodiment, E, G of 12,13 are replaced into the stronger A of hydrophobicity, to improve the anchoring ability of MHCII.
In the present embodiment, the T2 sequence after modification is following SEQIDNO:4 aminoacid sequence: AKFVKAWTLKLAA (SEQIDNO:4).T2 sequence derives from the cd4 t cell epi-position (GenBank:ABV58843.2, a.a59-71) of the hemagglutinin of influenza A virus (H3N8), and its aminoacid sequence is PKYVKQNTLKLAT (SEQIDNO:2).Similar to the modification of T1, inventor modifies as follows to PKYVKQNTLKLAT, to improve the ability of its induction Tfh differentiation:
The amino acid P of (1) the 1st is the anchor amino acids of MHCII, is generally hydrophobic amino acid.In the present embodiment, it is by for being replaced into the stronger A of hydrophobicity.
The amino acid Y of (2) the 3rd is the binding site of MHCII, and the F being all die aromatischen Aminosaeuren has stronger MHCII binding ability than Y.In the present embodiment, the Y of the 3rd is replaced into F.
The amino acid Q of (3) the 6th is the anchor amino acids of MHCII.In order to improve the anchoring ability of MHCII, the Q of the 6th is replaced into the stronger A of hydrophobicity.
The amino acid N of (4) the 7th is the binding site of TCR, and usual positively charged or aromatic amino acid can promote the combination with TCR.In the present embodiment, it is by for being replaced into aromatic amino acid W.
The amino acid T of (5) the 13rd is the anchor amino acids of MHCII.In the present embodiment, the T of 13 is replaced into the stronger A of hydrophobicity, to improve the anchoring ability of MHCII.
The synthesis of embodiment 2, polypeptide
With the peptide in chemical synthesis synthetic table 1.The present invention utilizes Merrifield solid phase synthesis process; use the automatic Peptide synthesizer of ABI company 433A type; adopt the amino acid of WANG resin and 9-fluorenylmethyloxycarbonyl (Fmoc amido protecting group) and the modification of other blocking group, according to from carboxyl terminal (-COOH) to aminoterminal (-NH
2) direction, the successively sequence of synthetic peptide in synthetic table 1.After synthesis completes, according to ABI company Peptide systhesis Standard cleavage program, application hydrofluoric acid cutting polypeptide and resin, makes polypeptide and resin isolation, simultaneously the blocking group of also deaminize acid side chain.
The cleaved polypeptide got off, by repetitive scrubbing and precipitation, carries out quantitative analysis by RPLC and mass spectrum.After the peptide masses analyzed is qualified, packing of weighing, then tests for downstream.
The emulsification of embodiment 3, polypeptide
Sequence number selected in precise table 1 is SEQIDNO:5, SEQIDNO:6, SEQIDNO:7, SEQIDNO:8 and SEQIDNO:9 totally 5 polypeptide (see table 1) being combined into peptide based immunogens, dissolve with DMSO (DMSO) and water for injection, after ultrasound filtration, be mixed with the polypeptide solution that concentration is 500 μ g/mL.The company of Total France white oil (pharmaceutical grade) measuring same volume, as adjuvant, is emulsified into water in oil synthetic peptide based immunogens with Britain's IKA mulser.Prepare water for injection and Dao Daer white-oil adjuvant emulsification group that one group does not contain any antigenic substance in addition, use as negative control.By after quality test for animal immune.
Table 1, PCV virus epitopes and array mode thereof
Embodiment 4, pig circular ring virus epi-position capsid protein (GenBank:ACZ06596.1, capsid [Porcinecircovirus-2], aa119-130 and aa231-233)
1, immunization protocol
SEQIDNO:5, SEQIDNO:6, SEQIDNO:7, SEQIDNO:8, the SEQIDNO:9 good according to above-mentioned emulsification amount to 5 kinds of synthetic peptide based immunogens, use often kind of synthetic peptide based immunogens immunity 5 mouse under the same conditions, the test immunization protocol immune mouse according to listing below:
Experimental group and size of animal: be divided into 6 groups, test group 5 groups, control group 1 group.
Laboratory animal: C57 mouse, often organizes 5 mouse.
Animal age, sex and body weight: 6 week age male mice, body weight 20 grams to 22 grams.
Synthetic peptide based immunogens adjuvant and formulation: Dao Daer white-oil adjuvant, water-in-oil.
Immunizing dose: every 0.2mL, every milliliter containing 500 μ g synthetic peptide based immunogens.
Blood sampling time: first 0th day of immunity, the 7th day, the 15th day and blood sampling in the 30th day after immunity.
Route of inoculation: two hind leg intramuscularly, point 2 injections, often some 0.1mL.
Perform in strict accordance with above-mentioned mouse animal experiment scheme.Be used for ELISA (enzyme linked immunosorbent assay) experiment at blood sampling separation of serum storage, determine Serum Antibody level.
2, indirect ELISA method measures mice serum antibody horizontal and result judgement
A. TPPA.With previously prepd sequence number SEQIDNOs:5-9 synthetic peptide bag by the 96 flat enzyme plates in hole, by indirect ELISA method, determine the antibody horizontal that mouse produces.Dissolve synthetic peptide SEQIDNOs:5-9 with the 0.05M carbonate buffer solution of PH=9.6, concentration is 7 μ g/mL, and every hole adds 100ul.4 degree of placements 16 hours.Close with 5% skim-milk (sigma)/PBS, 37 degree of insulations 3 hours.At once for indirect ELISA analysis after sealase target.
The mice serum of separation is first done 50 times of dilutions, then 3 times of serial dilutions are until the 8th row hole, and first row i.e. 50 times of dilutions, and serum-dilution is in table 2.Every hole adds sample 100 μ l, sets negative control simultaneously.Hatch 1 hour.After washing, add ELIAS secondary antibody, every hole 100 μ l, hatches 1 hour.Add tmb substrate after washing and react 15 minutes, add stop buffer.The OD value in each hole of enzyme plate is measured at wavelength 450nm.
Table 2, serum samples diluted multiple table
Note: X1 to X96 is the concrete sample OD value measured.
Result criterion: based on the linear regression analysis of optical density, is set as 0.5 by the cutoff that wavelength 450nm absorbs, and this set(ting)value is correct and accurate, because carry out each OD value analyzing dilution normal control mice sample be all less than 0.15.We use Reed-Muench method to measure the extension rate of each group of mouse serum sample when OD value.
Reed-Muench method formula:
Detected sample=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 0.5
X2 is lower than the diluted sample multiple OD value of cutoff 0.5
Calculate weighted mean afterwards, net result is in table 3.If serum samples diluted degree is higher, illustrate that immune effect is better.
Antibody test result after table 3, PCV vaccine 5 kinds of peptide based immunogens immune mouses, extension rate table
A represents lower than 50 times of dilutions, is less than cutoff 0.5 in this dilution range, negative.Therefore do not process.
B. result judges.The composition of visible T1-B-T2 and T2-B-T1 of mean OD value of mouse is often organized significantly better than other each group through indirect ELISA mensuration.
As can be seen from Figure 1, the result of mensuration can reflect the height of antibody horizontal intuitively, and the effect that also can embody t helper cell epi-position is strong and weak.Sequence SEQIDNO:5 does not produce antibody, shows if antigen does not have t helper cell epi-position to cause antibody response; The antibody dilution multiple that SEQIDNO:8 and SEQIDNO:9 two synthetic peptide combinations produce is more than 6 to 8 times of SEQIDNO:6 and SEQIDNO:7 composition.T cell epitope is directly connected to the generation of antibody, and good t cell epitope design can produce high antibody response.
Pass through interpretation of result, as can be seen from Figure 1, synthetic peptide SEQIDNO:5 epi-position group, combined peptide T1-B group SEQIDNO:6 and B-T2 group SEQIDNO:7 mean OD value, all well below the mean OD value of T1-B-T2 group SEQIDNO:8 and T2-B-T1SEQIDNO:9, illustrate that these two kinds of combined peptides of T1-B-T2 and T2-B-T1 have best immune effect.
3, flow cytometry is used to evaluate quantity and the frequency of Tfh cell
Asepticly adopt the mouse spleen of immunity after 8 days, add the erythrocyte cracked liquid of 2mL, squeeze out spleen cell with 5mL syringe afterbody, it is resuspended to add 5mL2% serum RPMI-1640 nutrient solution, and 1800 leave the heart 6 minutes, abandon supernatant.Cell precipitation is resuspended with 2% serum RPMI-1640 nutrient solution.After cell counting, with antibody (CD4, CD44, the CXCR5) labeled cell of various anti-Tfh cell marker molecules deriving from BD company, according to the BD application program operation dyeing of standard, then use BD flow cytometer CantoII measurement result.
Result criterion: Tfh quantity in the frequency that the Tfh cell measuring every mouse is shared in cd4 t cell and every spleen.
(1) frequency of different cd4 t cell and B cell epi-position combination induction Tfh: containing the synthetic peptide immune mouse after 8 days of different cd4 t cell and the combination of B cell epi-position, get spleen, remove red corpuscle, the full cell of separating spleen, adopts the fluorescent-labeled antibody for CD4, CD44, CXCR5 to dye.Tfh cell is defined as the cell mass of CD4+CD44+CXCR5+.Numerical value in Fig. 2 above rectangle is shown as the frequency of Tfh cell.Frequency is higher, illustrates that the immune effect of synthetic peptide is better.As can be seen from Figure 2, the frequency of T1-B-T2 group SEQIDNO:8 and T2-B-T1SEQIDNO:9, far away higher than the frequency of synthetic peptide SEQIDNO:5 epi-position group, combined peptide T1-B group SEQIDNO:6 and B-T2 group SEQIDNO:7, illustrates that these two kinds of combined peptides of T1-B-T2 and T2-B-T1 have best immune effect.
(2) quantity of different cd4 t cell and B cell epi-position combination induction Tfh: according to the frequency of cell and the quantity of the full cell of spleen, calculate the quantity of Tfh in each mouse spleen as follows: Tfh cell quantity=splenocyte quantity × cd4 t cell frequency × CD44+CXCR5+ cell frequencies.Quantity illustrates that the immune effect of synthetic peptide is better more at most.As can be seen from Figure 3, sequence SEQIDNO:5 does not produce antibody, shows if antigen does not have t helper cell epi-position to cause antibody response.T cell epitope is directly connected to the generation of antibody, and good t cell epitope design can produce high antibody response.The frequency of T1-B-T2 group SEQIDNO:8 and T2-B-T1SEQIDNO:9, far away higher than the quantity of synthetic peptide SEQIDNO:5 epi-position group, combined peptide T1-B group SEQIDNO:6 and B-T2 group SEQIDNO:7, illustrates that these two kinds of combined peptides of T1-B-T2 and T2-B-T1 have best immune effect.
Those skilled in the art should understand, although for illustrative purposes, this document describes the specific embodiment of the present invention, can carry out various amendment and without departing from the spirit and scope of the present invention to it.Therefore, the specific embodiment of the present invention and embodiment should not be considered as limiting the scope of the invention.The present invention is only by the restriction of claims.The all documents quoted in the application are all intactly incorporated to herein as a reference.
Claims (17)
1. a peptide species, it comprises the aminoacid sequence of T1-B-T2 or T2-B-T1 configuration arrangement,
Wherein, T1 is the SEQIDNO:1 after modifying;
B is SEQIDNO:5;
T2 is the SEQIDNO:2 after modifying.
2. polypeptide as claimed in claim 1, wherein, described modification comprises displacement, adds and/or lacks the one or more amino acid in original aminoacid sequence, and aminoacid sequence after modification and described original aminoacid sequence have identical function.
3. polypeptide as claimed in claim 2, wherein, the modification of described SEQIDNO:1 comprises following one or more modifications:
(1) the 1st amino acids Serine is hydrophobic amino acid;
(2) the 2nd amino acids L-glutamic acid are replaced into the amino acid of positively charged;
(3) the 12nd amino acids L-glutamic acid are replaced into hydrophobic amino acid; With
(4) the 13rd amino acids glycine are replaced into hydrophobic amino acid.
4. polypeptide as claimed in claim 2 or claim 3, wherein, the modification of described SEQIDNO:2 comprises following one or more modifications:
(1) the 1st amino acids proline(Pro) is replaced into hydrophobic amino acid;
(2) the 3rd amino acids tyrosine substitution are other die aromatischen Aminosaeurens;
(3) the 6th amino acids glutamine are replaced into hydrophobic amino acid;
(4) the 7th amino acids l-asparagines are replaced into positively charged or aromatic amino acid; With
(5) the 13rd amino acids Threonines are replaced into hydrophobic amino acid.
5. polypeptide as claimed in claim 3, wherein, T1 aminoacid sequence comprises the aminoacid sequence shown in SEQIDNO:3.
6. the polypeptide as described in claim 4 or 5, wherein, T2 aminoacid sequence comprises the aminoacid sequence shown in SEQIDNO:4.
7. the polypeptide as described in any one of claim 1-6, it is by synthetic, by protokaryon or eukaryotic cell expression or embed pathogenic agent by gene recombination technology and obtain.
8. the polynucleotide be separated, it comprises the one be selected from lower group:
A) many nucleic acids of the polypeptide of coding as described in any one of claim 1-6; Or
B) with the polynucleotide of polynucleotide a) complementary or stingent hybridization.
9. a vaccine, it comprises the polypeptide of at least one as described in claim 1-6.
10. vaccine as claimed in claim 9, it also comprises medical science or veterinarily acceptable delivery vector or adjuvant.
11. vaccines as described in claim 9 or 10, its route of administration is selected from intraocular, nose, intramuscular, injection, oral, intraperitoneal, subcutaneous, locally, intracutaneous and transdermal delivery.
12. 1 kinds of vaccine adjuvants, it comprises the polypeptide of at least one as described in claim 1-6.
13. polypeptide as described in any one of claim 1-6 are for the preparation of the purposes in the preventative vaccine of resisting porcine circovirus, therapeutic vaccine.
14. polypeptide as described in any one of claim 1-6 are preparing the purposes in mono-clonal or polyclonal antibody.
15. polynucleotide as claimed in claim 8 are preparing the purposes in polypeptide.
16. vaccines as described in any one of claim 9-11 are for the preparation of the purposes in the preventative vaccine of resisting porcine circovirus, therapeutic vaccine.
17. vaccine adjuvants as claimed in claim 12 are for the preparation of the purposes in the preventative vaccine of resisting porcine circovirus, therapeutic vaccine.
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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 |
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