CN104628865A - Pseudorabies virus epitope polypeptide gene engineering vaccine - Google Patents

Abstract

The invention relates to preparation and application of a Pseudorabies virus (PRV) epitope polypeptide gene engineering vaccine. The vaccine uses PRV as the main glycoprotein, and B cell neutralizing epitope of gB, gC and gD and T cell immune epitope as the vaccine frame structure. The vaccine is prepared by the following steps: connecting the main glycoprotein with the vaccine frame structure through a flexible linker, connecting in series with a molecular adjuvant Bovine Herpesvirus 1 (BHV-1) envelope protein VP22, cloning a pRSETB vector, transforming Escherichia coli, fermenting, purifying, emulsifying and the like to obtain the PRV epitope polypeptide gene engineering vaccine. The invention also relates to an application method of the vaccine. The animal experiment indicates that the PRV epitope polypeptide gene engineering vaccine has equivalent effect to the live virus vaccine in the aspect of humoral immunity and cellular immunity level, and can be stimulated to generate T lymphopoiesis immunoreaction on the cellular level and generate antibody immunoreaction with virus neutralizing activity on the humoral level.

Description

A kind of pseudo-rabies epitope polypeptide recombinant vaccine

Technical field

The invention belongs to biotechnology genetically engineered field, relate generally to the preparation and application of a kind of pseudo-rabies (Pseudorabies virus, PrV) epitope polypeptide recombinant vaccine.Particularly, utilize gene recombination technology, by the B cell neutralizing epitope of PRV primary glycoproteins gB, gC, gD and T cell immune epitope and bovine herpes virus-1 (Bovine Herpesvirus 1, BHV-1) envelope protein VP22 connects, and be cloned into carrier, transform Host Strains, by fermentation, prepared by purifying, emulsifying process, obtain pseudo-rabies epitope polypeptide recombinant vaccine and the application of this vaccine in the great Animal diseases pseudoabies of prevention.

Background technology

Pseudoabies is that one betides in domestic animal, wild mammal, companion animals and laboratory animal, with heating, very to itch (except pig) and encephalomyelitis is a kind of acute infectious disease of cardinal symptom, caused by pseudorabies virus (Pseudorabies virus, PrV).The genomic dna of pseudorabies virus is wire double-strand, be about 150kb, be made up of the terminal repeat (TR) of the long section of uniqueness (UL), unique short section (US), short section both sides and internal repeat (IR).Genomic dna at least codified 70 genes of Pseudorabies virus, wherein many genes are nonessential by disappearance virulence gene and cause weak, and its huge genome, can hold the insertion of larger foreign gene.

In UL district by the gene totally 58 kinds of locating, comprise be sequenced encode glycoproteins gB (gII), gC (gIII), gH, gK, gL, gM, gN, TK, alkaline nuclease (AN), ribonucleoside reductase enzyme (RR), DNA polymerase (POL), DBP (136kDa DNA binding protein dna), MCP (major capsid protein), the gene such as ICP18.5 albumen and early protein O (EPO).US district is all checked order, comprising the gene of encoding protein kinase (PK), glycoprotein gG (gX), gp50 (gD), gp63 (gI) and gI (gE) and the gene of coding 11kDa and 28kDa.The early gene (IE) in IR district and RSp40 gene have also been obtained qualification and order-checking, and prove that the IE gene between different strain there are differences.The latent gene of IR and UL joining region is also identified in addition.In 9 kinds of existing known PrV envelope glycoproteins (gX, gp50, gp63, gI, gII, gIII, gH, gM and gL) except gX, other is the constituent of ripe virion, only have gX to be non-constituent, be often present in cells infected, virion does not find.Up to the present, confirmed that gI, gIII, gp63, gX and gM are nonessential to virosome internal breeding, and gII, gp50, gH and gL by virus replication required.

The gE single-gene deletion of vaccine of current widespread use in use oneself its limitation of knocking gradually, be in particular in: after the immunity of gE deletion of vaccine, although produce clinical symptom after wild virus infection can be reduced and wild malicious discharge after infecting, but there is the pig of high maternal antibody as bred pigs or growing and fattening pigs, effectively neutralizing antibody can not be excited after using gE vaccine, thus virus more (Gielkens L J, 1984 are discharged after infection; Molitor T W etal., 1992), therefore sow needs repeated inoculation, and advise that market pig is in the immunity of 8,12,16 weeks difference once.The disappearance of gE causes virus to be bred in vivo very soon simultaneously, especially in scavenger cell, medullary cell and lymphocyte, although gE gene-deleted strain virulence declines to some extent, may produce immunosuppression to immune special close preferendum.GG deletion of vaccine compares aspect with other vaccine, also has different conclusions: no matter whether maternal antibody exists, gX and gE deletion of vaccine is in induction NAT and equal indifference (Boeker M, etal., 1999 in toxin expelling after preventing infections; Pensaert M B, De Waele K, 1990), Vamier P etal (1991).GG and gE is lacked seedling soluble in water respectively, after gG (gX) immunity, the antibody horizontal of induction lacks seedling a little more than gE.RzihaHJetal (1989) compares TK/gE and gE/TK two kinds of deletion mutantion strains propagation in vivo.Result illustrates: gE/TK 6 weeks after infection, after immunosuppressor process pig, the DNA of this gene-deleted strain can be detected from many tissues, and TK/gE only breeds in nervous tissue and tonsilla, and with extremely low quantity propagation, after immunosuppressor process pig, infective virus particle can not be detected, the early stage result of study of this result support (Kit etal., 1985), namely the vaccine of TK gene negative can reduce the ability that virus forms latent infection.

Although the protective immunity produced for PrV virus has made large quantifier elimination, the mechanism produced for protection and the definite character of antigen have still had the factor of many unknowns, and great majority research all highlights the importance of cellular immunization.But the mechanism of Prv virus immunity response is comparatively complicated, passive immunization can only spread by blocking virus from the epithelial cell of initial propagation, but can not stop virus copying and breeding at inoculation position.Eliminate from the epithelial cell of expressing high-level major histocompatibility complex (MHC) I class antigen and infect, by cytotoxic T lymphocyte, must eliminate to infect then depending on antibody in the neurocyte of not expressing MHC.Antibody plays certain effect in protection, but the dependency between antibody titers and clinical protection power is incomplete, because some negative antibody pig part when attacking poison obtains protection (Andries etal, 1978; McCawand Xu, 1993).

Have been found that PRV has 11 kinds of glycoprotein at present, wherein, gB, gC, gD are stimulates body to produce the albumen of neutralizing antibody, the antibody produced be no matter in vivo, external, or deposit with or without complement have in case in and the ability of PRV.Therefore, gB, gC, gD are the first-selected glycoprotein of development PRV subunit vaccine.B cell epi-position (Ober, B.T., etal, 1998 are detected in PRVgB, gC glycoprotein; K.H.Wiesmuller, etal, 2000; Zaripov, M.M., etal, 1998) also detect that gC Protein T cell epi-position can elicit humoral immune and cell-cytotoxic reaction (Ober, B.T., etal, 2000; ).PRV gD induces strong neutralizing antibody and faint cell-cytotoxic reaction.The immune parameter that (van Rooij, E.M., etal, 2000) fully understand protection relevant is the prerequisite improving anti-PRV virus vaccines.Based on important protective effect mechanism (Bianchi, A.T., 1998 that Th1 type Cell regulate is anti-PRV infection; Fischer, L., 2003; Van Rooij, E.M., 2004; ).Therefore, have the immune response of report immune induction Th1 preference that available protecting can be provided to resist fatal PRV to infect.(Yoon, H.A.etal, 2007). in these three primary glycoproteins, the plasmid DNA intramuscular injection immune animal of expressing gB albumen can induce the immune response of Th1 preference, provides the protection that the most effective anti-mortality attacks poison subsequently.(Yoon, H.A.etal, 2006). and the immunomodulating cytokines in Th1 preference immune response source be combined with the resistibility helping to strengthen anti-mortality PRV and infect.(Yoon，H.A.etal，2006；Yoon，H.A.etal，2007)。Sizable effect (Kimman, T.G., etal, 1992 have been played in specificity virus and in the control infected at control PRV of serum antibody; Marchioli, C., R.etal, 1988).PRV protective antigen gene gD is inserted into the downstream of human cytomegalic inclusion disease virus promotor by Marchioli etc.; then proceed in Chinese hamster ovary cell (CHO); the CHO gD-17 cell strain that gD albumen is expressed in 1 strain is filtered out through aminopterinum; adjuvant is equipped with expression product after mass propgation; immune mouse can produce the antibody of higher titre; attack poison after immune 3 times, mouse can resist strong virus attack.Intranasal vaccination immune swine, can induce pig to produce neutralizing antibody and protect pig to resist the attack of the strong poison of PRV.The gC that Katayama etc. (1991) are purified with heparin affinity chromatography and with gB, gC, gD Pigs Inoculated respectively that immunoaffinity chromatography is purified, all can make it resist the attack of PRV.The PRV glycoprotein gC of baculovirus expression can produce neutralizing antibody by inducing mouse.The PrV envelope protein gC of extraction is made immunogenic mixture and subunit vaccine by Tulman etc.All can induction of antibodies response and lymphproliferation response with subunit vaccine immune mouse and pig.With this subunit vaccine immune mouse and pig, all can induction of antibodies response and lymphproliferation response.With this subunit vaccine immunity twice, mouse can be protected from the attack of the strong poison of PRV of lethal quantity.Confirm that several protein herpesvirus is the target antigen of T cell or antibody, such as, the extremely early stage glycoprotein (Ballksetal., 1991) of herpes simplex virus I-type (HSV-1) specificity horse cytotoxic lymphocyte identification.Therefore, cells infected is just cleaved before infectious virus produces.Late period, Hsv-1 glycoprotein gB, gC and gD was the target antigen of people, horse, pig cell cytotoxic T cell, also oneself is considered to the target antigen (Ben-Poratetal of neutralizing antibody, 1986), antibody adsorption test shows that pig has produced the antibody for gE, pole early protein, nucleocapsid protein.It is the main antibody composition playing neutralizing effect in rehabilitation porcine blood serum that Zuckermann F A (1999) demonstrates gC antibody; GC glycoprotein is also the target antigen of the cellular immunization that produces of the thin pig of target of cytotoxicity CD8+ and CD4+T cell and immune swine and humoral immunization.GC glycoprotein two antigenic determinants of helper T cell identification are proved.Therefore gC is natural sense metachromia, does not rely on the effect of complement.GD glycoprotein is also the target antigen of T cell.GG can not stimulate generation neutralizing antibody (Thomsenetal1987), because gG is not the integral part of virus envelope.With gC or gD immune swine and the mouse of purifying, more can produce better protecting power than gG immunity, resist high virulence PRV strain and attack.The monoclonal anti physical efficiency passive protection mouse of gB, gC and gD and pig are for the lethal infection of PRV, and gE monoclonal antibody only can protect mouse.GE contains several antibody combining site; need complement could neutralize virus; gE also containing can by the epitope of helper T cell identification, but fail to prove that cytotoxic lymphocyte (CTL) identifies gE in mouse, also can protect mouse with the eucaryon of gE or prokaryotic expression product immunized mice.Pseudorabies virus gC glycoprotein is the target antigen of mouse and pig CTLS, and the function of gC is multiple: in the attachment of PRV, play keying action, and is main immunogen.This protein has the antibody of neutralizing effect at small white mouse and pig Immune inducing in vivo when complement exists, and is the target cell of t cell response;

Epitope polypeptide vaccine can design by the same antigen conservative region of different strains selected of specific aim; epitope information containing wide spectrum; get rid of the inhibition epi-position, pathology, the tolerance that exist in totivirus albumen and cause the unfavorable factor such as epi-position of different malicious strain difference; Cross immunogenicity between different strain is provided, and then successfully manages host's autogene and to suddenly change the immune evasion problem caused.Epiposition vaccine has also possessed the advantage not available for traditional vaccine technology, and it has broken away from the restriction of vitro culture, does not need live virus to participate in production, does not comprise the complete component of causal organism, has thoroughly broken away from potential threat, avirulence atavism, safer; Synthetic or engineering are expressed, and quality is more stable; The heredity restriction of MHC can be overcome and obtain efficient submission, autoimmune response or immunosuppression can not be caused; Molecular structure is little and simple, seldom there will be serious anaphylaxis and nosocomial infection problem; There is operability, carry out combined modification or the transformation of Different Kinds of Pathogens multi-epitope on a molecular scale.

Bovine herpes virus-1 (Bovine Herpesvims 1, BHV-1) envelope protein VP22 is protein transduction associated protein, there is unique protein transduction (Harms J S et al, 2000), the foreign protein merged with it direct transmembrane transport under mediating without any subsidiary conditions can be entered cell, and can at intercellular trafficking, and entered intracellular foreign protein by transduction and still retain its original biological activity (Wills K N et al., 2001, Elliott G O, 1997, Kretz A, Wybranietz WA, Hermening S et al., 2003).Therefore it can significantly improve offering efficiency and then improving its immunogenicity of foreign protein.Although the cross-film mechanism so far for protein transduction is explained not yet completely, still extensively carried out about the applied basic research of VP22 protein transduction characteristic, and obtained challenging achievement in research.Existing many sections of bibliographical informations confirm the immunological effect (Oliveira SC et al, 2001, Hung C F et al.2004) VP22 and target antigen gene fusion expression significantly being strengthened DNA vaccination at present.Existing many sections of research reports confirm the immunological effect (Oliveira S C et al, 2001, Hung CF et al.2004) VP22 and target antigen gene fusion expression significantly being strengthened DNA vaccination at present.Therefore by goal gene and VP22 amalgamation and expression be during recombinant vaccine designs new approaches.

Summary of the invention

The present invention is according to current domestic Major Epidemic strain glycoprotein gB, gC, gD aminoacid sequence, relevant biological information software DNASTAR, BIMAS and SYFPEITHI is utilized to analyze the secondary structure that it carries out wetting ability, antigenicity, plasticity-, surperficial accessibility and Gamier-Robson, again according to the conservative property design oligonucleotides fragment of B cell neutralizing epitope and t cell epitope position and aminoacid sequence, introduce bovine herpes virus I type envelope protein VP22 as adjuvant molecules simultaneously.By after designed Pseudorabies virus neutralizing epitope and the series connection of BVP22 molecular polypeptide in intestinal bacteria coexpression, by fermentation, purifying, the technique such as emulsification, obtain the pseudo-rabies epitope polypeptide recombinant vaccine with Desirable immunogenic.The vaccine utilizing the present invention to prepare effectively can prevent pseudoabies.

An object of the present invention there are provided a kind of new epitope polypeptide vaccine that can be used for preventing Pseudorabies virus epidemic isolates to infect and vaccine composition thereof.

Two of object of the present invention there are provided structure and the preparation method of described epitope polypeptide vaccine.

Three of object of the present invention there are provided the engineering strain can expressing described pseudo-rabies epitope polypeptide vaccine.

Four of object of the present invention there are provided the preparation method of described pseudo-rabies epitope polypeptide vaccine.

Five of object of the present invention there are provided the purposes of described epitope polypeptide vaccine in prevention pseudoabies.

In first aspect, the invention provides a kind of its composition of epitope polypeptide vaccine amino acid infected for pseudo-rabies epidemic isolates.It contains 3 primary glycoproteins gB, gC, gD B cell neutralizing epitopes, the t cell epitope of gB, gC glycoprotein and bovine herpes virus envelope protein VP22 molecule adjuvant.Described epitope polypeptide vaccine comprises gB, gC, gD many B cell and t cell epitope and molecule adjuvant and to be connected in series together formed albumen or polypeptide or pharmacy acceptable salt and coexpression epitope and the carrier required for molecule adjuvant polypeptide protein.Carrier also can comprise the sequence of each epitope of coding separately, or independent coding molecule adjuvant BVP22 binding sequence.Series connection can be undertaken by genetic engineering method, except containing 3 primary glycoproteins gB, gC, gD B cell neutralizing epitopes in protein engineering vaccine, outside the t cell epitope of gB, gC glycoprotein and bovine herpes virus envelope protein VP22 molecule adjuvant, also comprise nonimmune active substance.Described nonimmune active substance is the connection portion of each polypeptide, does not have the immunogenicity of epitope, does not also have any adjuvanticity, mainly contains purification tag, joint peptide, chemically modified part, N end signal peptide and C and holds polyadenylic acid etc.Described pharmacy acceptable salt refers to nontoxicity, stimulation and transformation reactions, is applicable to the salt of human or animal tissues.Inactive substance and pharmacy acceptable salt are well known to those skilled in the art.

Primary glycoproteins epitope sequence preferably described in the polyepitope vaccines polypeptide of first aspect present invention is from Major Epidemic strain primary glycoproteins gB, gC, gD B cell neutralizing epitope and t cell epitope, and molecule adjuvant is bovine herpes virus envelope protein VP22.

Preferred in addition, described in first aspect present invention, the aminoacid sequence of epitope polypeptide vaccine is as follows:

(1) gB Glycoprotein B cell antigen epitope 1:

NRFTDRVPVPVQEITDVIDRRGKCVSKAEYVRNNHKVTAFDRDENPVEVDLRPSRLNALGTRGWHTTNDTYTKIG

(2) gB Glycoprotein B cell antigen epitope 2:

APGRFQQVEHYYPIDLDSRLRASESVTRNFLRTPHFTVAWDWAPKTRRVCSLAKWREAEEMIRDETRDGSF；

(3) gB glycoprotein T cell antigen epi-position 1:

ISRLRRNPMKALYPVTTKALKEDGVEEDDVDEAKLDQARDMIRYMSIVSALEQQEHKARKKNSGPALLASRVGAMATRRRHYQRLESEDPDAL

(4) gC Glycoprotein B cell antigen epitope 1:

TPRVPPPSVSRRKPQRNGNRTRVHGDEATSHGR

(5) gC Glycoprotein B cell antigen epitope 2:GRFRSPDADPEYFDEPPRPELPRE

(6) gC glycoprotein T cell antigen epi-position 1:DYYPRRSV;

(7) gC glycoprotein T cell antigen epi-position 2:DTQRYDAS;

(8) gD Glycoprotein B cell antigen epitope 1:FYQQPPHREVVNYWYRKNGRTLP

(9) gD Glycoprotein B cell antigen epitope 2:GSPRPRPRPRPRPSPRPKPEPAP

(10) gD Glycoprotein B cell antigen epitope 3:RPTPRPPRPETPHRPF

(11) molecule adjuvant is bovine herpes virus envelope protein VP22:

FHRPSEDEDDYEYSDLWVRENSLYDYESGSDDHVYEELRAATSGPEPSGRGGPARRSSSRASSRPGGDSDPPKSNERLGGSRLRGERARP

In second aspect, the invention provides a kind of nucleic acid molecule, the pseudo-rabies epitope polypeptide vaccine of its coding described in first aspect present invention.Nucleotide of the present invention can be rna form, DNA form, many epitopes tandem sequence and molecule adjuvant sequence is synthesized by synthetic mode, then connect rear clone through genetically engineered operation and enter carrier, be transformed into intestinal bacteria, after screening, fermentation, purifying, obtain epitope polypeptide vaccine protein.Conventional molecular biology manipulations can be carried out in the present invention to this nucleic acid, as: PCR, digestion with restriction enzyme, connect, and nucleic acid design 5 ' end and 3 ' end all add restriction enzyme site.Nucleotide sequence in preferred the present invention is as follows:

GGATCCTTCCACAGGCCCTCCGAAGACGAGGACGATTACGAGTACAGCGACCTTTGGGTGCGAGAAAACAGCCTCTATGACTACGAGTCCGGCTCGGATGACCACGTATACGAAGAGCTGCGCGCCGCGACGAGCGGACCCGAGCCGAGCGGGCGGGGTGGTCCGGCCCGCCGCTCGAGCAGCCGGGCGTCCTCGCGCCCGGGTGGTGACCCCCCAAAGAGCAACGAGCGATTGGATCGCGGTGGTAGCCGCCTTCGCGGCGAGCGGGCCCGGCCGGGTGATATCCCAGGTTGCAAGACGCCCCGGGTCCCGCCGCCCTCGGTCTCGCGCCGGAAGCCCCAGCGGAACGGCAACAGGACGCGCGTCCACGGCGACGAGGCCACCTCGCACGGGCGCGGTGGTGGGCGCTTCCGCTCGCCCGACGCCGACCCCGAGTACTTTGACGAGCCCCCGCGCCCGGAGCTCCCGCGGGAGGGTGGTGACTACTACCCGCGGCGCAGCGTGGGTGGTACGCAGCGCTACGACGCCTCCCCCGGTTCTGGTAACCGCTTCACGGACCGCGTGCCCGTCCCCGTGCAGGAGATCACGGACGTGATCGACCGCCGCGGCAAGTGCGTCTCCAAGGCCGAGTACGTGCGCAACAACCACAAGGTGACCGCCTTCGACCGCGACGAGAACCCCGTCGAGGTGGACCTGCGCCCCTCGCGCCTGAACGCGCTCGGCACCCGCGGCTGGCACACCACCAACGACACCTACACCAAGATCGGCGGTGGTGCGCCCGGGCGCTTCCAGCAGGTGGAGCACTACTACCCCATCGACCTGGACTCGCGCCTCCGCGCCTCCGAGAGCGTGACGCGCAACTTTCTGCGCACGCCGCACTTCACGGTGGCCTGGGACTGGGCCCCCAAGACGCGGCGCGTGTGCAGCCTGGCCAAGTGGCGCGAGGCCGAGGAGATGATCCGCGACGAGACGCGCGACGGGTCCTTCGGTGGTATCTCGCGCCTGCGCCGCAACCCCATGAAGGCCCTGTACCCCGTCACGACGAAGGCGCTCAAGGAGGACGGCGTCGAAGAGGACGACGTGGACGAGGCCAAGCTGGACCAGGCCCGGGACATGATCCGGTACATGTCCATCGTGTCGGCCCTCGAGCAGCAGGAGCACAAGGCGCGCAAGAAGAACAGCGGGCCCGCGCTGCTGGCCAGCCGCGTCGGGGCGATGGCCACGCGCCGCCGGCACTACCAGCGCCTCGAGAGCGAGGACCCCGACGCCCTGGGTTCTGGTTTCTACCAGCAGCCCCCGCACCGGGAGGTGGTGAACTACTGGTACCGCAAGAACGGCCGGACGCTCCCGGGTGGTGGCTCGCCGAGGCCCAGGCCCAGGCCCAGGCCCAGGCCCAGCCCCCGGCCGAAGCCCGAGCCCGCCCCGGGTGGTCGCCCCACGCCGCGACCCCCGAGGCCCGAGACGCCGCACCGCCCCTTCTAAGCTT

In the third aspect, the invention provides a kind of carrier, it is except the nucleic acid molecule containing the coding pseudo-rabies epitope polypeptide vaccine described in second aspect present invention, also containing being connected with this nucleotide sequence is exercisable, at the expression controlling elements needed for procaryotic cell expression (transcribe and translate).The most basic expression controlling elements comprises promotor, transcription terminator, enhanser, selected marker etc., and these controlling elements are known in the art.In a preferred embodiment, described expression vector is coli expression carrier.

In fourth aspect, the invention provides a kind of host cell, it contains the carrier described in third aspect present invention.Host cell through to transform or transfection contains the gene order of proteins encoded of the present invention, after then there is good Inheritance and expression stability after testing, can be used for fermentation expression produce needed for pseudo-rabies epitope polypeptide vaccine protein.

In the 5th, the invention provides a kind of preparation method of pseudo-rabies epitope polypeptide vaccine, it comprises the following steps: engineering bacterium fermentation expresses epitope polypeptide vaccine protein, through thick purifying and polishing purification technique and follow-up emulsifying process, and the epitope polypeptide vaccine required for acquisition.The method wherein related to includes, but are not limited to bacterial cell disruption, inclusion body washing, centrifugal, sex change, affinity chromatography, hydrophobic chromatography, anion-exchange chromatography, reverse-phase chromatography, renaturation, emulsification etc.The preparation method related in the present invention is well known to those skilled in the art.

In the 6th, the invention provides a kind of for preventing the epitope polypeptide vaccine of pseudoabies, it comprises polypeptide described in first aspect present invention and pharmaceutically acceptable carrier.Described pseudo-rabies vaccine can prevent infecting of pseudo-rabies epidemic isolates.Pharmaceutically acceptable carrier of the present invention is immunostimulant or immunological adjuvant, and preferred immunological adjuvant is import white-oil adjuvant.

In the 7th, the invention provides the application of the pseudo-rabies epitope polypeptide vaccine described in the 6th aspect.Vaccine can necessarily effective dose intramuscular injection, and intracutaneous or subcutaneous injection or intranasal vaccination injection animal, neutralizing antibody and the cytokine (as IFN etc.) that can produce q.s provide antiviral activity, watch for animals.In addition, in embodiments of the invention, by carrying out target animals immunity simultaneous test, laboratory safety test etc. to vaccine, show that pseudo-rabies epitope polypeptide recombinant vaccine of the present invention is safe, pseudo-rabies epitope polypeptide recombinant vaccine effect in humoral immunization and cellular immune level is suitable with living virus vaccine, can stimulate on a cellular level and produce the immune response of T lymphopoiesis, body fluid levels produce the antibody mediated immunity reaction with viral Neutralization effect.

In addition, it is pointed out that on the basis of the contextual disclosure of the application, the aspect that other have substantive distinguishing features of the present invention is apparent concerning the ordinary skill people of this area.In addition, the present invention which also uses open source literature, and their entire contents is all included in and carried out reference herein.

Accompanying drawing explanation

Following accompanying drawing for illustration of specific embodiment of the invention scheme, and is not used in the scope of the invention limiting and defined by claims.

Fig. 1 is expression vector pRSETB-PRV (the gB/gC/gD)-BVP22 schematic diagram of pseudo-rabies epitope polypeptide recombinant vaccine encoding gene.Fig. 2 is digestion with restriction enzyme qualification electrophorogram.1:DL2000DNAMarker; 2: empty vector control; 3:pRSETB-PRV (gB/gC/gD)-BVP22 plasmid double digestion; 4:pRSETB-PRV (gB/gC/gD)-BVP22 plasmid.Fig. 3 is bacterial classification abduction delivering SDS-PAGE electrophorogram.1: molecular weight of albumen Marker:97.2KD, 66.4KD, 44.3KD, 29.0KD, 20.1KD, 14.3KD; 2-3 abduction delivering (for the purpose of arrow indication albumen); 4: the contrast bacterium of non-abduction delivering.Fig. 4 is Sample Purification on Single Western Blot Blot results.1: pre-dyed Marker; 2: sample; 3: negative control.Fig. 5 is fermented sample SDSPAGE electrophorogram.1: molecular weight of albumen Marker:97.2KD, 66.4KD, 44.3KD, 29.0KD, 20.1KD, 14.3KD; 2: do not express contrast; 3: fermented sample.Fig. 6 is fermentation purifying protein Western Blot Blot results.1: pre-dyed molecular weight of albumen Marker; 2: fermentation purification of samples; 3: blank.Fig. 7 is that immune serum ELISA detects gC glycoprotein specific antibodies detected result; Fig. 8 is that PRV stimulates hamster kidney cell T lymphproliferation response detected result; Fig. 9 is that PRV stimulates PBMC T lymphproliferation response detected result.

Embodiment

It is only exemplary description that concrete test method described in embodiment describes, and for elaborating the present invention, but does not form limitation of the scope of the invention, to be manyly changed to known by those skilled in the art according to of the present invention.

The mentality of designing of embodiment one pseudo-rabies epitope polypeptide vaccine protein

The present invention is according to current domestic pseudo-rabies Major Epidemic strain glycoprotein gB, gC, gD aminoacid sequence, relevant biological information software DNASTAR, BIMAS and SYFPEITHI is utilized to analyze the secondary structure that it carries out wetting ability, antigenicity, plasticity-, surperficial accessibility and Garnier-Robson, again according to the conservative property design oligonucleotides fragment of B cell neutralizing epitope and t cell epitope position and aminoacid sequence, introduce bovine herpes virus I type envelope protein VP22 as adjuvant molecules simultaneously.By after designed Pseudorabies virus B cell and t cell epitope and the series connection of BVP22 molecular polypeptide in intestinal bacteria coexpression, by fermentation, purifying, the technique such as emulsification, obtain the pseudo-rabies epitope polypeptide vaccine with Desirable immunogenic.The vaccine utilizing the present invention to prepare effectively can prevent pseudoabies.

The genome sequences such as comprehensive analysis domestic Pseudorabies virus epidemic strain SA, Bartha, HNQX, ZJNB, HuNXT, antigenic structure, Advance of Epidemiological Research, be optimized design to restructuring pseudo-rabies epitope polypeptide vaccine.The present invention utilizes bioinformatics software to its glycoprotein gB, gC, the wetting ability that gD carries out, antigenicity, plasticity-, the secondary structure of surface accessibility and Gamier-Robson is analyzed, predict on the basis of possible B cell antigen epi-position and t cell epitope, according to the similarity of epitope position and aminoacid sequence, analyze each epidemic isolates and have epitope, and with reference to the sequence information in GenBank, the epitope of prediction is compared, the conservative property of further analysis epitope in different virus strain, thus determine and gB Glycoprotein B cell dominant antigen epitope polypeptide 2 sections, t cell epitope 1 section, gC Glycoprotein B cell epitope 2 sections, t cell epitope 2 sections, gD Glycoprotein B cell epitope 3 sections.By forming the skeleton structure of vaccine after all epi-position series connection, add molecule adjuvant at skeleton nitrogen end simultaneously.The overall structure of this vaccine is:

Molecular Adjuvant(BVP22)-gC B Cell Epitope 1-gC B Cell Epitope 2-gC T Cell Epitope 1-gC T Cell Epitope 2-gB B Cell Epitope 1-gB B Cell Epitope 2-gB T Cell Epitope 1-gD B Cell Epitope 1-gD B Cell Epitope 2-gD B Cell Epitope 3

The structure of embodiment two coli expression carrier and expression strain

The polypeptide-coding nucleotide designed in embodiment one is served the synthesis of extra large handsome biotech company, nucleotide fragments two ends devise BamH I (5 ' end) and HindIII (3 ' end) restriction enzyme site respectively, being cloned on pMD18T carrier respectively after these 2 segment condenses, sequencing confirms to insert gene fragment consistent with implementation sequence (see sequence table).By recombinant plasmid called after pMD18T-PRV (gB/gC/gD)-BVP22, with corresponding restriction enzyme, two kinds of plasmids are carried out enzyme and cut process, coli expression carrier selects the pRSETB plasmid of Invitrogen company, also identical restriction enzyme ferment treatment is used, enzyme tangent condition: 10 μ l reaction systems, 2 μ l plasmids are added in system, restriction enzyme is 5 activity units (New England biolabs), add 10 × damping fluid 1 μ l, deionized water polishing, 37 DEG C of enzymes cut 1.5 hours.Enzyme adds 1 μ l 200mM EDTA termination reaction after cutting end.In 1% agarose gel electrophoresis, electrophoresis 30 minutes.Under ultraviolet lamp, PRV (the gB/gC/gD)-BVP22 fragment of 2.9kb pRSETB plasmid, 1480bp is cut, reclaim test kit specification sheets according to Qiagen company gel and carry out glue recovery.According to carrier: ratio independent and expression vector mixing by multi-epitope nucleotide fragments of fragment 1: 2-3, reaction system 15 μ l, connected by T4DNA ligase enzyme, 16 DEG C of connections are spent the night, obtain recombinant plasmid called after pRSETB-PRV (gB/gC/gD)-BVP22 (see Fig. 1), transform competent E. coli BL21 (DE3) pLysS.

Transform: pRSETB-PRV (gB/gC/gD)-BVP22 is put thawed on ice, add 2 μ l Ligature liquid, again mix, ice-water bath 30 minutes, 42 DEG C 30 seconds, then ice bath is put back to rapidly 1.5 minutes, add 1ml LB nutrient solution, 37 DEG C, quiescent culture 1 hour, 4000g low-temperature centrifugation 10 abandons supernatant second, with the resuspended thalline of 200 μ lLB substratum; Bacterium liquid is spread evenly across on the LB agar plate containing 100 μ g/mL penbritins, is inverted in 37 DEG C of thermostat containers and cultivates 12-16 hour, until Clone formation.

Qualification: the mono-clonal on picking flat board is in LB substratum, 37 DEG C, 200rpm shakes cultivation 12 hours, extract plasmid, restriction endonuclease BamH I and HindIII is used to carry out double digestion, can cut out the clone of corresponding pseudorabies vaccines gene size fragment, 1480bp, tentatively can be defined as positive colony (see Fig. 2); Positive colony carries out determined dna sequence and verifies its exactness (see sequence table) further.

Abduction delivering.By positive colony incubated overnight, morning next day, by 1: 100 switching, cultivates after 3 hours, adds 0.5mM IPTG, continues cultivation 3 hours, prepares sample; Conventional SDS-PAGE testing goal protein expression situation---at 56KD molecular weight place (see Fig. 3), see specific band for correct clone; Get correct clone, amplification culture, SDS-PAGE confirms to express correctly, and it expresses accuracy (see Fig. 4) to use conventional Western-blot to confirm further; After above-mentioned structure and qualification program, the positive colony selected can be carried out the foundation of original species word bank as engineering bacteria, bacterial classification called after pRSETB-PRV (gB/gC/gD)-BVP22/BL21 (DE3, Plys).

The fermentation of embodiment three engineering bacteria, purifying and emulsification

Production bacterial classification is got in fermentation, is inoculated in (containing 100 μ g/ml penbritins) in 2ml LB liquid nutrient medium, 37 DEG C, 180rpm shaking culture 12 hours activated spawn.Access shaking flask with the inoculum size of 1: 100 again, 37 DEG C of shaking culture, to OD600=3, can be inoculated into fermentor tank in 10% ratio.Fermentation substratum is semisynthetic medium, with distilled water preparation, wherein not containing any microbiotic.Correct dissolved oxygen and pH value electrode, open tank body and stir, revolution is 300rpm, the online sterilizing of tank body, when culture-liquid temp is down to 37.0 DEG C in tank, demarcates pH and dissolved oxygen (OD) zero point.Leavening temperature is 37.0 ± 0.1 DEG C, dissolved oxygen controls about 20%, pH controls 7.0, flow feeding 500ml during cultivation thalline OD600=1.0 ~ 1.2 after inoculation, within after feed supplement 1 hour, add IPTG (final concentration is 0.5mM) abduction delivering, continuous induction secondary fermentation in 6 hours terminates, and sampling is SDS-PAGE and is examined and determine expression.

The thalline that purifying will be collected, ultrasonic with carrying out after occlusion body washing lotion I (1%Triton X-100,20mMTris-cl PH8.0) suspendible, 2000W ultrasonic degradation 1 hour.4 DEG C, 12000rpm collected by centrifugation occlusion body, and wash occlusion body with occlusion body washing lotion II (1%DOC, 4M urea, 20mMTris-cl PH8.0) suspendible twice ultrasonic, secondary low-temperature centrifugation collects occlusion body.Occlusion body precipitation uses 8M urea, and 0.3% β-ME, 20mM Tris-cl (pH=8.00) mix, and stirring at room temperature 4 hours, 8000rpm low-temperature centrifugation 30min, discards precipitation.Metaprotein 1: 100 dilutes, and renaturation solution Tris (PH8.0) buffer system, adds 0.3M arginine, and 4 DEG C are stirred renaturation 24 hours.The 20mM phosphoric acid buffer of renaturation solution pH=8.0,0.5M sodium-chlor, 20mM imidazoles, affinity column in balance, with the 20mM phosphoric acid buffer of pH=8.0,0.5M sodium-chlor, 0.5M imidazoles wash-out; Use 1.5M (NH again ₄) ₂the 10mM Na of SO4,100mMEDTA, pH=8.5 ₂hPO ₄hydrophobic chromatography post in balance, reequilibrate, with the 10mM Na of pH=8.5 ₂hPO ₄wash-out, obtains pseudo-rabies polypeptide epitope vaccine work in-process stoste.Do whether SDS-PAGE and Western blot marking calibrating purified product is target protein (Fig. 5, Fig. 6).

The PBS of the work in-process sterilizing of purifying is diluted to 200 μ g/ml by emulsification.Get import white mineral oil adjuvant DUOPRIME (pharmaceutical grade) through 121 DEG C, sterilizing 15 minutes, for subsequent use.By oil phase: aqueous phase=50: the proportions of 50, first oil phase is added in emulsion tank, start stirrer and slowly stir with the speed of 80-100r/min, slowly add aqueous phase, stir 2min again after adding, then with 5500r/min high-speed circulating emulsification 9min, make the single-phase vaccine of water-in-oil.

Embodiment four pseudo-rabies epitope polypeptide vaccine safety is tested

Material

Vaccine: pseudo-rabies epitope polypeptide vaccine, lot number is 20120604,20120608,20120612.

Experimental animal (1) 18-22g small white mouse 17, purchased from medicine inspecting institute of Qingdao City.(2) pregnant sow 8,5 week age piglet 12, it is negative that IDEXX ELISA commercial kit detects PRV, and Guangdong Winsun Bio-Pharmaceutical Co., Ltd.'s experimental animal center provides, and pig is starting to test front environmental adaptation one week.

Method

Vaccine is to the security of small white mouse

Subcutaneous injection small white mouse, often only injects 0.5ml, often criticizes vaccination 5, injects 15 small white mouses altogether, sets 2 negative controls, Continuous Observation 10 days simultaneously, the healthy state of observation small white mouse.

Vaccine is to the security of pregnant sow

6 female Landrace are pregnant 40 ~ 60 days time, and intramuscular injection lmL pseudo-rabies epitope polypeptide vaccine respectively, every batch of immune two, establishes 2 sows as non-immunized controls in addition, observes farrowing situation.

Vaccine is to the security of piglet

The epitope polypeptide vaccine that PRV negative antibody piglet 9 provides for injection center, often criticizes vaccination 3 pigs, and every pig posterior auricular muscle meat is injected 1ml and amounted to 2ml.Set up negative control 3, every injection white-oil adjuvant 2ml, clinical observation 14 days simultaneously.Off-test, every pig is weighed.

Result

Vaccine is to the safety testing of small white mouse

Result is as table 1, and 20120612 immune group 1 animal subjects, second day body temperature slightly raises, and within the 3rd day, recovers normal, appetite and healthy state without exception, consistent with control group, occur without dead, visible pseudo-rabies epitope polypeptide vaccine is safe to small white mouse, in table 1.

Table 1 vaccine is to the safety testing result of small white mouse

group	size of animal	body temperature	appetite	healthy state	dead quantity
						20120604	5	1 fervescence, other are normal.	normally	be in a good state of health	0
20120608	5	normally	normally	be in a good state of health	0
						20120612	5	normally	normally	be in a good state of health	0
contrast	2	normally	normally	be in a good state of health	0

Vaccine is to the safety testing of pregnant sow

Show that test sow is raised to a point puerperium by table 2 result, apart from individual other weak tire and stillborn foetus, do not occur miscarriage, premature labor and product mummy tire phenomenon, prove that this vaccine is safe and reliable to pregnant sow, the production performance of pregnant sow is not affected.Duration of test vaccine group and control group test pig all do not occur that abnormal clinical reacts, and spirit, search for food normal, and injection site does not have inflammatory symptom.

Table 2 farrowing sow production performance result

Vaccine is to the safety testing of piglet

Result is as table 3, vaccine group and control group test pig all do not occur that abnormal clinical reacts, spirit, search for food normal, injection site does not have inflammatory symptom, body temperature is all normal, this vaccine group of immune group piglet average weight gain is respectively 2.5Kg, 2.4Kg, blank group average weight gain 2.3Kg, does not present significant difference (P > 0.05) between group.Refer to table 1.In whole 14 days experimental observation phases, all immunity totally 95 week age piglet, body temperature and appetite are all normal, do not occur any clinical unusual phenomenon, and after off-test, 9 piglets are all strong to live, with control group indifference.This illustrates, pseudo-rabies epitope polypeptide vaccine is safe to piglet.

Table 3 piglet security detected result

The grouping of embodiment five animal and immunity

In order to the efficiency evaluation parameter obtaining vaccine as much as possible, the present invention have detected the relevant T lymphopoiesis situation of the specific antibody after immune balb/c mice, the virucidin of mouse and piglet and cellular immunization.

1 vaccine is with to attack poison viral

Pseudo-rabies epitope polypeptide vaccine is provided by Hongqiao Ming Qin research and development centre, lot number is 20120604,20120608,20120612, gE gene nature deletion of vaccine strain (Bartha-K61) living vaccine is so kind as to give by pharmaceutical development center, Yongshun, Guangdong, and lot number is 2012016.

2 experimental animals and grouping

The BalB/C female mice in 25 5 week ages, is divided into 5 groups at random, epitope polypeptide vaccine three lot number groups, living vaccine group and control group, often organizes 5/cage; 25 5 ~ 6 week age piglet, male and female half and half, are divided into 5 groups immediately, epitope polypeptide vaccine three lot number groups, living vaccine group and control group, often organize 5, experiment before experimental animal raise one week in advance, conform.

3 test design and method

Carry out immunity according to grouping situation to Balb/C mouse and piglet, after head exempts from, two weeks booster immunizations once, 20 μ g/ or 20 μ g/ heads.Head exempts from latter 2 weeks, 4 weeks, 6 weeks, 8 weeks, mouse docking blood sampling also separation of serum, and after last blood sampling, slaughters mouse, is separated hamster kidney cell and is used for T lymphocyte proliferation assay; Head exempts from latter 6 weeks, 8 weeks, 10 weeks, and piglet precaval vein blood sampling also separation of serum and PBMC is used for cellular immunization detection.

Embodiment six indirect ELISA detects gC antibody response

The CBS (PH9.6) of recombinant protein purification sample 50mmol/l is diluted to 1 μ g/ml, adds 96 hole elisa plates, every hole 100 μ l, 4 DEG C of bag quilts that spend the night.After PBST washing lotion buffer washs three times, with confining liquid 5% horse serum (PBS buffer system) in 37 DEG C, close 1 hour.After washing three times, mouse serum sample confining liquid (5% horse serum, PBS buffer system) is diluted to 1: 20, and every hole adds 100 μ l, and two repetitions, hatch 1 hour for 37 DEG C; After washing three times, every hole adds the HRP-mark two anti-(sheep anti mouse) being diluted to 1: 10000, hatches 1 hour for 37 DEG C; After washing three times, every hole adds 50 μ l tmb substrates, and room temperature, lucifuge, reaction 10min, add 2M H ₂sO ₄stopped reaction.Light absorption value (BIORAD 680 microplate reader) is read under 450nm wavelength.

Result

Result

As shown in Figure 7, different group produces the gC protein specific antibody of different levels.Whole duration of test, epitope polypeptide vaccine and the seedling group mouse that lives create obvious gC protein-specific IgG antibody, are pole significant difference (P < 0.01) with contrasting.Result shows that epitope polypeptide vaccine provided by the invention can stimulate Balb/C mouse to produce humoral immune reaction, and antibody horizontal is suitable with existing living vaccine, without significant difference (P > 0.05).

Embodiment seven antibody neutralization test

The serum that after exempting from head, 4,6,8 weeks gather, carries out virucidin (SN) and detects.Blood sample carries out 56 DEG C of deactivation 30min before carrying out serum Neutralizing test, adds the 100TCID of equivalent after twice serial dilution ₅₀pRV Bartha-K61 strain, adds 96 well culture plates, 5%CO ₂incubator (Sanyo MCO-15AC), cultivate 1 hour for 37 DEG C, then, add 100 μ l Vero cell suspensions, every hole contains 2 × 10 ⁴individual cell.Culture plate is placed in 5%CO ₂moist environment, cultivate 3 days for 37 DEG C, read to survey the cytopathy of viral dilution, each number infecting hole is expressed as the ratio that every viral dilution inoculates each hole count.In and titre be as the criterion with the most highly diluted multiple not observing CPE.Each sample does three repetitions.

Result

In table 4, after head exempts from, 4 weeks (head exempts from rear 2 weeks booster immunizations) detects neutralizing antibody first in all immune group.Whole duration of test, the neutralizing antibody that restructuring ubiquitination vaccine group and Attenuate vaccine group produce is on close level, but immune group is apparently higher than the neutralizing antibody of control group, presents pole significant difference (P < 0.01); Exempt from latter 8 weeks to head, control group does not produce any neutralizing antibody.

Table 4 immune mouse neutralizing antibody detects

Note: 1 antibody titers < 1: 8 is considered as without neutralizing antibody; 2NR represents antibody titers without definite result; 3a represents the quantity of mouse

Embodiment eight lymphopoiesis detects

In order to contrast cell-mediated immune reaction further, within 8 weeks after head exempts from, carrying out cell proliferation test, after head exempts from, within 6,8,10 weeks, having carried out Swine PBMC proliferation test, to detect the cell immune response of vaccine immunity.Hamster kidney cell or piglet PBMC are put into 96 porocyte plates, 100 μ l/ holes (2 × 10 ⁵cells/well).Add 100 μ l/ substratum subsequently, or add the PRV through uv irradiating deactivation, mixing.Concanavalin A (5 μ g/ml, Sigma) is as positive control.Nephrocyte or PBMC sample do three repetitions.According to the method that Bounous provides, carry out hamster kidney cell or Swine PBMC proliferation activity by the MTT colorimetry improved to detect: hamster kidney cell 72 hours, or after PBMC cultivates 96 hours, every hole adds 10 μ l WST, hatch 5 hours, after hatching under 490nm wavelength reading.Stimulation index (SI) mean value in antigen stimulated cells hole calculates than the mean value in cell (stimulation) hole.

Result

With neutralizing antibody response class seemingly, the renal cell proliferation observing highest level in epitope polypeptide vaccine group and living vaccine group is active, sees Fig. 8, Fig. 9.The T lymphopoiesis of control group is starkly lower than other immune group (P < 0.05), the stimulation index of epitope polypeptide vaccine group and living vaccine group is close, does not present significant difference (P > 0.05).Similar with hamster kidney cell, latter 6 weeks, 8 weeks, 10 weeks of immunity, have also discovered this rule (Fig. 9) in detecting to Swine PBMC propagation.These data show that epitope polypeptide vaccine can strengthen cell immune response at natural host Immune inducing in vivo.

The detection of embodiment nine vaccine immunity piglet cell immune response

Method is shown in embodiment seven, the results are shown in Table 5.Head exempts from latter 6 weeks, 8 weeks, 10 weeks, gathers serum sample and is used for virus neutralization tests.Whole duration of test, control group piglet sample does not detect PRV neutralizing antibody, and this is consistent with the result of hamster kidney cell.All immune group detect neutralizing antibody in 6 weeks after head exempts from, and after immunity, 8 weeks, 10 weeks epi-position polypeptide vaccine groups and the whole immune piglet of living vaccine group detect neutralizing antibody.Epitope polypeptide vaccine group has four-head piglet NAT to reach 1: 32, and living virus vaccine group has one to reach 1: 32; Living vaccine group after immunity 10 weeks, have a pig NAT to be 1: 8, other immune piglets all reach 1: 16.

The immune piglet neutralizing antibody of table 5 detects

Note: 1 antibody titers < 1: 8 is considered as without neutralizing antibody; 2NR representative is without definite result; 3a represents the quantity of pig.

Claims (7)

1. a pseudo-rabies epitope polypeptide vaccine fusion rotein, its aminoacid sequence is SEQ ID No.2.

2. a nucleic acid molecule, its coding claim 1 described fusion rotein.

3. a carrier, it contains nucleic acid molecule according to claim 2.

4. a host cell, it contains carrier according to claim 3.

5. nucleic acid molecule according to claim 2, its concrete sequence is as follows:

gga tcc ttc cac agg ccc tcc gaa gac gag gac gat tac gag tac agc gac ctt tgg gtg cga gaa aac agc ctctat gac tac gag tcc ggc tcg gat gac cac gta tac gaa gag ctg cgc gcc gcg acg agc gga ccc gag ccgagc ggg cgg ggt ggt ccg gcc cgc cgc tcg agc agc cgg gcg tcc tcg cgc ccg ggt ggt gac ccc cca aagagc aac gag cga ttg gat cgc ggt ggt agc cgc ctt cgc ggc gag cgg gcc cgg ccg ggt gat atc cca ggttgc aag acg ccc cgg gtc ccg ccg ccc tcg gtc tcg cgc cgg aag ccc cag cgg aac ggc aac agg acg cgcgtc cac ggc gac gag gcc acc tcg cac ggg cgc ggt ggt ggg cgc ttc cgc tcg ccc gac gcc gac ccc gagtac ttt gac gag ccc ccg cgc ccg gag ctc ccg cgg gag ggt ggt gac tac tac ccg cgg cgc agc gtg ggtggt acg cag cgc tac gac gcc tcc ccc ggt tct ggt aac cgc ttc acg gac cgc gtg ccc gtc ccc gtg cag gagatc acg gac gtg atc gac cgc cgc ggc aag tgc gtc tcc aag gcc gag tac gtg cgc aac aac cac aag gtgacc gcc ttc gac cgc gac gag aac ccc gtc gag gtg gac ctg cgc ccc tcg cgc ctg aac gcg ctc ggc acccgc ggc tgg cac acc acc aac gac acc tac acc aag atc ggc ggt ggt gcg ccc ggg cgc ttc cag cag gtggag cac tac tac ccc atc gac ctg gac tcg cgc ctc cgc gcc tcc gag agc gtg acg cgc aac ttt ctg cgc acgccg cac ttc acg gtg gcc tgg gac tgg gcc ccc aag acg cgg cgc gtg tgc agc ctg gcc aag tgg cgc gaggcc gag gag atg atc cgc gac gag acg cgc gac ggg tcc ttc ggt ggt atc tcg cgc ctg cgc cgc aac cccatg aag gcc ctg tac ccc gtc acg acg aag gcg ctc aag gag gac ggc gtc gaa gag gac gac gtg gac gaggcc aag ctg gac cag gcc cgg gac atg atc cgg tac atg tcc atc gtg tcg gcc ctc gag cag cag gag cacaag gcg cgc aag aag aac agc ggg ccc gcg ctg ctg gcc agc cgc gtc ggg gcg atg gcc acg cgc cgccgg cac tac cag cgc ctc gag agc gag gac ccc gac gcc ctg ggt tct ggt ttc tac cag cag ccc ccg caccgg gag gtg gtg aac tac tgg tac cgc aag aac ggc cgg acg ctc ccg ggt ggt ggc tcg ccg agg ccc aggccc agg ccc agg ccc agg ccc agc ccc cgg ccg aag ccc gag ccc gcc ccg ggt ggt cgc ccc acg ccgcga ccc ccg agg ccc gag acg ccg cac cgc ccc ttc taa gct t 。

6., for preventing a vaccine for pseudoabies, it comprises fusion rotein according to claim 1 and pharmaceutically acceptable carrier.

7. fusion rotein according to claim 1 is preparing the application in pseudo-rabies epitope polypeptide recombinant vaccine.