CN104628865B

CN104628865B - A kind of pseudo- mad dog epitope polypeptide recombinant vaccine

Info

Publication number: CN104628865B
Application number: CN201510009086.9A
Authority: CN
Inventors: 李殿明; 蒲勤; 张毓金; 齐春梅; 田春辉; 刘甜甜; 任百亮; 张导春; 党将将; 吴启凡
Original assignee: Qingdao Mingqin Biological Technology Co Ltd
Current assignee: QINGDAO MINGQIN BIOLOGICAL TECHNOLOGY CO., LTD.
Priority date: 2015-01-06
Filing date: 2015-01-06
Publication date: 2017-12-19
Anticipated expiration: 2035-01-06
Also published as: CN104628865A

Abstract

The present invention relates to a kind of preparation and application of pseudo- mad dog (Pseudorabies virus, PrV) epitope polypeptide recombinant vaccine.The vaccine is used as vaccine frame structure using PRV primary glycoproteins gB, gC, gD B cell neutralizing epitope and T cell immune epitope, by after flexible linker connections again with molecule adjuvant bovine herpes virus-1 (Bovine Herpesvirus 1, BHV 1) envelope protein VP22 series connection, Escherichia coli are converted after being cloned into pRSETB carriers, through techniques such as everfermentation, purifying, emulsifications, the pseudo- mad dog epitope polypeptide recombinant vaccine with Desirable immunogenic is obtained.The invention further relates to the application method of the vaccine.Zoopery shows, pseudo- mad dog epitope polypeptide recombinant vaccine is suitable with living virus vaccine in humoral immunity and effect on cellular immune level, it can on a cellular level stimulate and produce T lymphopoiesis immune responses, producing the antibody mediated immunity with viral neutralization activity on body fluid levels reacts.

Description

A kind of pseudo- mad dog epitope polypeptide recombinant vaccine

Technical field

The invention belongs to biotechnology genetic engineering field, relate generally to a kind of pseudo- mad dog (Pseudorabies virus, PrV) the preparation and application of epitope polypeptide recombinant vaccine.Specifically, using gene recombination technology, by PRV primary glycoproteins GB, gC, gD B cell neutralizing epitope and T cell immune epitope and bovine herpes virus-1 (Bovine Herpesvirus 1, BHV-1) envelope protein VP22 connects, and is cloned into carrier, converts Host Strains, and through everfermentation, prepared by purifying, emulsifying process, obtain To the application of pseudo- mad dog epitope polypeptide recombinant vaccine and the vaccine in great Animal diseases pseudoabies is prevented.

Background technology

Pseudoabies is that one kind is betided in domestic animal, wild mammal, companion animals and experimental animal, with heating, very Itch (except pig) and encephalomyelitis is a kind of acute infectious disease of cardinal symptom, be by pseudorabies virus Caused by (Pseudorabies virus, PrV).The genomic DNA of pseudorabies virus is wire double-strand, is about 150kb, by Unique long section (UL), unique short section (US), the terminal repeat (TR) and internal repeat (IR) of short section both sides Composition.The genomic DNA of Pseudorabies virus at least 70 genes of codified, many of which gene are non-required can to pass through missing Virulence gene and cause weak, and its huge genome, the insertion of larger foreign gene can be accommodated.

In totally 58 kinds of the gene that UL areas have been positioned, including be sequenced coding glycoprotein gB (gII), gC (gIII), GH, gK, gL, gM, gN, TK, alkaline nuclease (AN), ribonucleotide reductase (RR), DNA polymerases (POL), DBP (136kDa DNA binding protein dna), MCP (major capsid protein), the gene such as ICP18.5 albumen and early protein O (EPO).US areas are complete Portion is sequenced, including encoding protein kinase (PK), glycoprotein gG (gX), gp50 (gD), gp63 (gI) and gI (gE) gene And coding 11kDa and 28kDa gene.The early gene (IE) and RSp40 genes in IR areas are also identified and are sequenced, and are demonstrate,proved IE genes between bright different strains have differences.The latent gene of other IR and UL bonding pads has also been identified.Existing known 9 In kind PrV envelope glycoproteins (gX, gp50, gp63, gI, gII, gIII, gH, gM and gL) in addition to gX, other is ripe virus The constituent of son, only gX is non-constituent, is normally present in infection cell, is not found on virion.To being at present Only, it is non-required to virion internal breeding to have proven to gI, gIII, gp63, gX and gM, and gII, gp50, gH and gL are virus It is required to replicate institute.

The gE single-genes deletion of vaccine being widely used at present its limitation of oneself gradual knocking in use, specific manifestation ：After gE deletion of vaccine is immune, although the discharge of wild poison after clinical symptoms and infection is produced after reducing wild virus infection, In the pig with high maternal antibody such as bred pigs or growing and fattening pigs, using can not effectively excite neutralizing antibody after gE vaccines, thus After infection discharge virus it is more (Gielkens L J, 1984；Molitor T W etal., 1992), therefore sow needs weight Reinoculation, and suggest that market pig was immunized once respectively at 8,12,16 weeks.GE missing causes virus to be bred in vivo very simultaneously It hurry up, especially in macrophage, bone marrow cell and lymphocyte, although gE gene-deleted strain virulence has declined, to exempting from The special close preferendum of epidemic disease system may produce immunosupress.The comparison aspect of gG deletion of vaccine and other vaccines, also has not Same conclusion：No matter maternal antibody presence or absence, gX and gE deletion of vaccine is in induction neutralizing antibody titers and prevents infections heel row Malicious aspect indifference (Boeker M, etal., 1999；Pensaert M B, De Waele K, 1990), Vamier P etal(1991).GG and gE missing seedling difference is soluble in water, and the antibody level induced after gG (gX) is immune is slightly above gE missings Seedling.RzihaHJetal (1989) compares the propagation of two kinds of deletion mutation strains of TK/gE and gE/TK in vivo.As a result illustrate：gE/ TK 6 weeks after infection, after handling pig with immunodepressant, the DNA of the gene-deleted strain can be detected from many tissues, and TK/gE is only Breed in nerve fiber and tonsillotome, and bred with extremely low quantity, after handling pig with immunodepressant, it is impossible to detect Infective virion, this result support early stage result of study (Kit etal., 1985), the i.e. vaccine of TK gene negatives The ability that virus forms latent infection can be reduced.

Although substantial amounts of research is made for protective immunity caused by PrV viruses, for mechanism caused by protection Still there are many unknown factors with the definite property of antigen, most of researchs all highlight the importance of cellular immunity.But Prv diseases The mechanism of malicious immune response is complex, passive immunity can only blocking virus spread from the epithelial cell initially bred, but not Duplication and propagation of the virus in inoculation position can be prevented.From high-level major histocompatibility complex (MHC) the I class antigens of expression Epithelial cell eliminate infection, it is necessary to by cytotoxic T lymphocyte, and in the nerve cell for not expressing MHC eliminate sense Dye then depends on antibody.Antibody plays certain effect in protection, but related between antibody titer and clinical protection power Property be incomplete because some negative antibody pigs when attacking poison part obtain protection (Andries etal, 1978；McCawand Xu, 1993).

Have now been found that PRV there are 11 kinds of glycoprotein, wherein, gB, gC, gD are the egg for stimulating body to produce neutralizing antibody In vain, caused antibody either in vivo, in vitro, or in having in the presence of whetheing there is complement and PRV ability. Therefore, gB, gC, gD are the preferred glycoprotein for developing PRV subunit vaccines.B cell table is detected in PRVgB, gC glycoprotein Position (Ober, B.T., etal, 1998；K.H.Wiesmuller, etal, 2000；Zaripov, M.M., etal, 1998) also detect To gC Protein T cells epitope can induction body fluid be immune and cell-cytotoxic reaction (Ober, B.T., etal, 2000；).PRV gD Induce strong neutralizing antibody and faint cell-cytotoxic reaction.(van Rooij, E.M., etal, 2000) is fully understood by protecting Related immune parameter is the premise for improving anti-PRV viral vaccines.It is the important guarantor of anti-PRV infection based on the regulation of Th1 types cell Protect effect mechanism (Bianchi, A.T., 1998；Fischer, L., 2003；Van Rooij, E.M., 2004；).Therefore, there is report Road immune induction Th1 preferences immune response can provide effectively protection and resist fatal PRV infection.(Yoon, H.A.etal, 2007) in these three primary glycoproteins, animal, which is immunized, in the DNA intramuscular injection of expression gB albumen can induce Th1 preferences Immune response, then provide the protection that maximally effective anti-mortality attacks poison.(Yoon, H.A.etal, 2006) and Th1 is inclined The combination of the immunomodulating cytokines in good immune response source helps to strengthen the resistance of anti-mortality PRV infection. (Yoon, H.A.etal, 2006；Yoon, H.A.etal, 2007).In specificity virus and serum antibody is in control PRV infection Control in played sizable effect (Kimman, T.G., etal, 1992；Marchioli, C., R.etal, 1988). PRV protective antigen genes gD is inserted into the downstream of human cytomegalovirus promoter by Marchioli etc., is then transferred to Chinese storehouse In mouse gonad cell (CHO), the CHO gD-17 cell lines of 1 plant of expression gD albumen are filtered out through ammonia petrin, are used after mass propgation Expression product is equipped with adjuvant, and immune mouse can produce the antibody compared with high titre, attacks poison after immune 3 times, mouse can resist strong poison and attack Hit.Intranasal vaccination immune swine, pig can be induced to produce neutralizing antibody and protect the attack of the strong poison of pig resistance PRV.Katayama etc. (1991) gC purified with heparin affinity chromatography and gB, gC, gD for being purified with immunoaffinity chromatography distinguish Pigs Inoculated, can make it Resist PRV attack.The PRV glycoprotein gC of baculovirus expression can inducing mouse generation neutralizing antibody.Tulman etc. will be extracted PrV envelope proteins gC immunogen compound i.e. subunit vaccine is made.Mouse and pig, which is immunized, with subunit vaccine can lure Lead antibody response and lymphproliferation response.Mouse and pig is immunized with this subunit vaccine, can induction of antibodies response and Lymphproliferation response.It is immune twice with this subunit vaccine, mouse attacking from the strong poison of PRV of lethal dose can be protected Hit.The target antigen that several protein herpesvirus are T cell or antibody is had proven to, for example, herpes simplex virus I-type (HSV-1) Specific horse cytotoxic lymphocyte identification pole early stage glycoprotein (Ballksetal., 1991).Therefore, infection cell is being felt Metachromia virus is just cleaved before producing.Late period Hsv-1 glycoprotein gB, gC and gD are people, horse, the target of pig cell cytotoxic T cell Antigen, also oneself is considered as the target antigen (Ben-Poratetal, 1986) of neutralizing antibody, and antibody adsorption test shows that pig has produced It is raw to be directed to gE, pole early protein, the antibody of nucleocapsid protein.It is rehabilitation pig that Zuckermann F A (1999), which demonstrate gC antibody, The main antibody composition of neutralization is played in serum；GC glycoprotein is also the thin pig of target of cytotoxicity CD8+ and CD4+T cell With immune swine caused by the target antigen of cellular immunity and humoral immunity.Two antigens of gC glycoprotein of helper T lymphocyte identification are determined Determine cluster to have been found.Therefore gC is natural sense metachromia, the effect independent of complement.GD glycoprotein is also the target antigen of T cell.gG It can not all stimulate and produce neutralizing antibody (Thomsenetal1987), because gG is not the part of virus envelope.With purifying GC or gD immune swines and mouse, it is more immune than gG more to produce preferable protection, resist high virulence PRV strains attack.GB, gC and GD monoclonal antibody energy passive protection mouse and pig for PRV lethal infection, and gE monoclonal antibodies be only capable of protect mouse.GE contains several Individual antibody combining site, it is necessary to complement could neutralize virus, gE also containing the epitope that can be identified by helper T lymphocyte, but It not can prove that cytotoxic lymphocyte (CTL) identifies to gE in mouse, can with gE eucaryon or prokaryotic expression product immunized mice yet Protect mouse.Pseudorabies virus gC glycoprotein is mouse and pig CTLS target antigen, and gC function is a variety of：In PRV attachment Middle performance key effect, and be main immunogene.The protein is in small white mouse and pig Immune inducing in vivo in the presence of complement Antibody with neutralization, and be the target cell of t cell response；

The same antigen conservative region for the different strains that epitope polypeptide vaccine can be directed to Sexual behavior mode is designed, and contains wide spectrum Epitope information, exclude inhibition epitope present in totivirus albumen, pathology, tolerance and cause different malicious strain differences The unfavorable factors such as epitope, there is provided the Cross immunogenicity between different strains, and then successfully manage host's autogene mutation institute Caused by immune evasion problem.Epiposition vaccine has been also equipped with the advantage not available for traditional vaccine technology, and it has broken away from external training Foster limitation, do not need live virus to participate in production, the complete component not comprising causal organism, thoroughly broken away from potential threat, Avirulence atavism, it is safer；Artificial synthesized or quantity sheet reaches, and quality is more stable；Can overcome MHC heredity limitation and Efficient submission is obtained, autoimmune response or immunosupress will not be caused；Molecular structure is small and simple, seldom occurs serious Allergic reaction and hospital-acquired infection problem；With operability, carry out repairing combining for Different Kinds of Pathogens multi-epitope on a molecular scale Decorations or transformation.

Bovine herpes virus-1 (Bovine Herpesvims 1, BHV-1) envelope protein VP22 is protein transduction correlation egg In vain, there is unique protein transduction (Harms J S et al, 2000), can be by the foreign protein merged therewith without appointing Direct cross-film is transported into cell under what subsidiary conditions mediation, and can enter into the cell in intercellular trafficking, and by transduction Foreign protein still retain its original bioactivity (Wills K N et al., 2001, Elliott G O, 1997, Kretz A, Wybranietz WA, Hermening S et al., 2003).Therefore it can significantly improve the efficiency of offering of foreign protein and enter And improve its immunogenicity.Although do not explained completely yet for the cross-film mechanism of protein transduction so far, on VP22 eggs White transduction properties applied basic research is still carried out extensively, and obtains encouraging achievement in research.Existing more texts at present Offering report confirms to significantly increase VP22 and target antigen gene fusion expression into immunological effect (the Oliveira SC et of DNA vaccination Al, 2001, Hung C F et al.2004).Existing more research report is confirmed VP22 and target antigen Gene Fusion table at present Up to the immunological effect (Oliveira S C et al, 2001, Hung CF et al.2004) for significantly increasing DNA vaccination.Therefore It is a new approaches in recombinant vaccine design by target gene and VP22 amalgamation and expressions.

Summary of the invention

The present invention utilizes relevant biological information according to current domestic Major Epidemic strain glycoprotein gB, gC, gD amino acid sequence Learn software DNASTAR, BIMAS and SYFPEITHI it is carried out hydrophily, antigenicity, plasticity, surface accessibility and Gamier-Robson secondary structure is analyzed, further according to B cell neutralizing epitope and t cell epitope position and amino acid sequence The conservative design oligonucleotides fragment of row, while bovine herpes virus I type envelope proteins VP22 is introduced as adjuvant molecules.By institute Co-expressed after design Pseudorabies virus neutralizing epitope and the series connection of BVP22 molecular polypeptides in Escherichia coli, through everfermentation, pure The techniques such as change, emulsification, obtain the pseudo- mad dog epitope polypeptide recombinant vaccine with Desirable immunogenic.Prepared using the present invention Vaccine can effectively prevent pseudoabies.

An object of the present invention is the provision of and a kind of new can be used to prevent Pseudorabies virus prevalence virus strain infection Epitope polypeptide vaccine and its vaccine combination.

The second object of the present invention is the provision of the structure and preparation method of the epitope polypeptide vaccine.

The third object of the present invention is the provision of the genetic engineering bacterium that can express the mad dog epitope polypeptide vaccine of the puppet Strain.

The fourth object of the present invention is the provision of the preparation method of the mad dog epitope polypeptide vaccine of the puppet.

The fifth object of the present invention is the provision of purposes of the epitope polypeptide vaccine in pseudoabies is prevented.

In a first aspect, the invention provides a kind of epitope polypeptide vaccine amino acid for being used for pseudo- mad dog prevalence virus strain infection Its composition.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 adjuvants.The epitope polypeptide vaccine includes the how individual B cell of gB, gC, gD and T cell table Position and molecule adjuvant be connected in series together formed albumen or polypeptide or pharmaceutically acceptable salt and coexpression antigen table Carrier required for position and molecule adjuvant polypeptide protein.Carrier can also include the sequence of separately encoded each epitope, or Separately encoded molecule adjuvant BVP22 binding sequences.Series connection can be carried out by genetic engineering method, in protein engineering vaccine except Contain 3 primary glycoproteins gB, gC, gD B cell neutralizing epitopes, the t cell epitope and bovine herpes virus of gB, gC glycoprotein Outside envelope protein VP22 molecule adjuvants, also comprising nonimmune active material.The nonimmune active material is the connection of each polypeptide Part, the immunogenicity without epitope, also without any adjuvanticity, mainly there are purification tag, joint peptide, chemistry Modified part, N-terminal signal peptide and C-terminal polyadenylic acid etc..The pharmaceutically acceptable salt refers to non-toxic, stimulation and metamorphosis Reaction, suitable for the salt of human or animal tissues.Inert matter and pharmaceutically acceptable salt are ripe for those skilled in the art Know.

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

In addition preferably, the amino acid sequence of epitope polypeptide vaccine is as follows described in first aspect present invention：

(1) gB Glycoprotein Bs cell antigen epitope 1：

NRFTDRVPVPVQEITDVIDRRGKCVSKAEYVRNNHKVTAFDRDENPVEVDLRPSRLNALGTRGWHTTND TYTKIG

(2) gB Glycoprotein Bs cell antigen epitope 2：

APGRFQQVEHYYPIDLDSRLRASESVTRNFLRTPHFTVAWDWAPKTRRVCSLAKWREAEEMIRDETRDGSF；

(3) gB glycoprotein T cell antigen epitope 1：

ISRLRRNPMKALYPVTTKALKEDGVEEDDVDEAKLDQARDMIRYMSIVSALEQQEHKARKKNSGPALLA SRVGAMATRRRHYQRLESEDPDAL

(4) gC Glycoprotein Bs cell antigen epitope 1：

TPRVPPPSVSRRKPQRNGNRTRVHGDEATSHGR

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

(6) gC glycoprotein T cell antigen epitope 1：DYYPRRSV；

(7) gC glycoprotein T cell antigen epitope 2：DTQRYDAS；

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

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

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

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

FHRPSEDEDDYEYSDLWVRENSLYDYESGSDDHVYEELRAATSGPEPSGRGGPARRSSSRASSRPGGDS DPPKSNERLGGSRLRGERARP

In second aspect, the invention provides a kind of nucleic acid molecule, and it encodes pseudo- mad described in first aspect present invention Dog epitope polypeptide vaccine.Nucleotide of the present invention can be rna form, DNA form, more antigens be synthesized by artificial synthesized mode Epitope tandem sequence and molecule adjuvant sequence, then operate connection rear clone by genetic engineering and enter carrier, be transformed into large intestine bar Bacterium, screening, fermentation, epitope polypeptide vaccine protein is obtained after purification.Conventional molecule can be carried out to the nucleic acid in the present invention Biologic operation, such as：PCR, digestion with restriction enzyme, connection etc., the end of nucleic acid design 5 ' and 3 ' ends add restriction enzyme site.It is excellent Nucleotide sequence in anthology invention is as follows：

GGATCCTTCCACAGGCCCTCCGAAGACGAGGACGATTACGAGTACAGCGACCTTTGGGTGCGAGAAAAC AGCCTCTATGACTACGAGTCCGGCTCGGATGACCACGTATACGAAGAGCTGCGCGCCGCGACGAGCGGACCCGAGCC GAGCGGGCGGGGTGGTCCGGCCCGCCGCTCGAGCAGCCGGGCGTCCTCGCGCCCGGGTGGTGACCCCCCAAAGAGCA ACGAGCGATTGGATCGCGGTGGTAGCCGCCTTCGCGGCGAGCGGGCCCGGCCGGGTGATATCCCAGGTTGCAAGACG CCCCGGGTCCCGCCGCCCTCGGTCTCGCGCCGGAAGCCCCAGCGGAACGGCAACAGGACGCGCGTCCACGGCGACGA GGCCACCTCGCACGGGCGCGGTGGTGGGCGCTTCCGCTCGCCCGACGCCGACCCCGAGTACTTTGACGAGCCCCCGC GCCCGGAGCTCCCGCGGGAGGGTGGTGACTACTACCCGCGGCGCAGCGTGGGTGGTACGCAGCGCTACGACGCCTCC CCCGGTTCTGGTAACCGCTTCACGGACCGCGTGCCCGTCCCCGTGCAGGAGATCACGGACGTGATCGACCGCCGCGG CAAGTGCGTCTCCAAGGCCGAGTACGTGCGCAACAACCACAAGGTGACCGCCTTCGACCGCGACGAGAACCCCGTCG AGGTGGACCTGCGCCCCTCGCGCCTGAACGCGCTCGGCACCCGCGGCTGGCACACCACCAACGACACCTACACCAAG ATCGGCGGTGGTGCGCCCGGGCGCTTCCAGCAGGTGGAGCACTACTACCCCATCGACCTGGACTCGCGCCTCCGCGC CTCCGAGAGCGTGACGCGCAACTTTCTGCGCACGCCGCACTTCACGGTGGCCTGGGACTGGGCCCCCAAGACGCGGC GCGTGTGCAGCCTGGCCAAGTGGCGCGAGGCCGAGGAGATGATCCGCGACGAGACGCGCGACGGGTCCTTCGGTGGT ATCTCGCGCCTGCGCCGCAACCCCATGAAGGCCCTGTACCCCGTCACGACGAAGGCGCTCAAGGAGGACGGCGTCGA AGAGGACGACGTGGACGAGGCCAAGCTGGACCAGGCCCGGGACATGATCCGGTACATGTCCATCGTGTCGGCCCTCG AGCAGCAGGAGCACAAGGCGCGCAAGAAGAACAGCGGGCCCGCGCTGCTGGCCAGCCGCGTCGGGGCGATGGCCACG CGCCGCCGGCACTACCAGCGCCTCGAGAGCGAGGACCCCGACGCCCTGGGTTCTGGTTTCTACCAGCAGCCCCCGCA CCGGGAGGTGGTGAACTACTGGTACCGCAAGAACGGCCGGACGCTCCCGGGTGGTGGCTCGCCGAGGCCCAGGCCCA GGCCCAGGCCCAGGCCCAGCCCCCGGCCGAAGCCCGAGCCCGCCCCGGGTGGTCGCCCCACGCCGCGACCCCCGAGG CCCGAGACGCCGCACCGCCCCTTCTAAGCTT

In the third aspect, the invention provides a kind of carrier, and it is except pseudo- containing the coding described in second aspect of the present invention The nucleic acid molecule of mad dog epitope polypeptide vaccine, also contain with the exercisable connection of the nucleotide sequence, in procaryotic cell expression Expression control element needed for (transcription and translation).Most basic expression control element includes promoter, transcription terminator, enhancing Son, selected marker etc., these controlling elements are known in the art.In a preferred embodiment, the expression vector is Coli expression carrier.

In fourth aspect, the invention provides a kind of host cell, and it contains the carrier described in third aspect present invention.Place Chief cell is inverted or transfects the gene order containing encoding proteins of the present invention, then has good heredity after testing After expression stability, available for the pseudo- mad dog epitope polypeptide vaccine protein needed for fermentation expression production.

At the 5th aspect, the invention provides a kind of preparation method of the mad dog epitope polypeptide vaccine of puppet, and it includes following step Suddenly：Engineering bacterium fermentation expresses epitope polypeptide vaccine protein, by thick purifying and polishing purification technique and follow-up emulsifying process, obtains Obtain required epitope polypeptide vaccine.The method being directed to include but is not limited to bacterial cell disruption, inclusion body washing, centrifugation, Denaturation, affinity chromatography, hydrophobic chromatography, anion-exchange chromatography, reverse-phase chromatography, renaturation, emulsification etc..The preparation being related in the present invention Method is well known to those skilled in the art.

At the 6th aspect, the invention provides a kind of epitope polypeptide vaccine for being used to prevent pseudoabies, it includes this hair Polypeptide and pharmaceutically acceptable carrier described in bright first aspect.The pseudo- rabies vaccine being capable of the pre- false proof popular poison of mad dog Strain is infected.Pharmaceutically acceptable carrier of the present invention is immunopotentiator or immunologic adjuvant, and preferably immunologic adjuvant is Import white-oil adjuvant.

At the 7th aspect, the invention provides the application of the pseudo- mad dog epitope polypeptide vaccine described in the 6th aspect.Vaccine can With certain effective dose intramuscular injection, intracutaneous or hypodermic injection or intranasal vaccination injection animal, the neutralization of sufficient amount can be produced Antibody and cell factor (such as IFN) provide antiviral activity, protect animal.In addition, in embodiments of the invention, pass through Target animals are carried out to vaccine contrast test, laboratory safety experiment etc. is immunized, show that the mad dog epitope of puppet of the present invention is more Peptide gene engineered vaccine is safe, and pseudo- mad dog epitope polypeptide recombinant vaccine is in humoral immunity with being imitated on cellular immune level Fruit is suitable with living virus vaccine, can on a cellular level stimulate and produce T lymphopoiesis immune responses, and tool is produced on body fluid levels The antibody mediated immunity for having viral neutralization activity reacts.

In addition, it is necessary to, it is noted that on the basis of the disclosure of the context of the application, other of the invention have The aspect of substantive distinguishing features is obvious for the ordinary skill people of this area.In addition, the present invention which also uses disclosure Document, their entire contents are included to be referred to herein.

Brief description of the drawings

Drawings below is used to illustrate specific embodiments of the present invention, rather than limits what is be defined by the claims The scope of the invention.

Fig. 1 is the expression vector pRSETB-PRV (gB/gC/ of pseudo- mad dog epitope polypeptide recombinant vaccine encoding gene GD)-BVP22 schematic diagrames.Fig. 2 is that digestion with restriction enzyme identifies electrophoretogram.1：DL2000DNAMarker；2：Empty carrier pair According to；3：PRSETB-PRV (gB/gC/gD)-BVP22 plasmid double digestions；4：PRSETB-PRV (gB/gC/gD)-BVP22 plasmids.Figure 3 be strain induced expression SDS-PAGE.1：Molecular weight of albumen Marker：97.2KD, 66.4KD, 44.3KD, 29.0KD, 20.1KD, 14.3KD；2-3 induced expressions (arrow meaning is purpose albumen)；4：The control bacterium of non-induced expression.Fig. 4 For Sample Purification on Single Western Blot Blot results.1：Pre-dyed Marker；2：Sample；3：Negative control.Fig. 5 is fermented sample SDSPAGE electrophoretograms.1：Molecular weight of albumen Marker：97.2KD, 66.4KD, 44.3KD, 29.0KD, 20.1KD, 14.3KD；2： Control is not expressed；3：Fermented sample.Fig. 6 is fermentation purifying protein Western Blot Blot results.1：Pre-dyed molecular weight of albumen Marker；2：Ferment purification of samples；3：Blank control.Fig. 7 is that immune serum ELISA detects gC glycoprotein specific antibodies Testing result；Fig. 8 is that PRV stimulates hamster kidney cell T lymphproliferation response testing results；Fig. 9 is that PRV stimulates PBMC T lymphs Cell proliferative response testing result.

Embodiment

Specific test method description described in embodiment is only exemplary description, for elaborating the present invention, but simultaneously It is not meant to limit the scope of the invention, many changes according to the present invention are well known to those skilled in the art.

The mentality of designing of the pseudo- mad dog epitope polypeptide vaccine protein of embodiment one

The present invention is raw using correlation according to pseudo- mad dog Major Epidemic strain glycoprotein gB, gC, gD amino acid sequence domestic at present Thing informatics software DNASTAR, BIMAS and SYFPEITHI it is carried out hydrophily, antigenicity, plasticity, surface accessibility and Garnier-Robson secondary structure is analyzed, further according to B cell neutralizing epitope and t cell epitope position and amino acid sequence The conservative design oligonucleotides fragment of row, while bovine herpes virus I type envelope proteins VP22 is introduced as adjuvant molecules.By institute Co-express, pass through in Escherichia coli after design Pseudorabies virus B cell and the series connection of t cell epitope and BVP22 molecular polypeptides The techniques such as fermentation, purifying, emulsification, obtain the pseudo- mad dog epitope polypeptide vaccine with Desirable immunogenic.Prepared using the present invention Vaccine can effectively prevent pseudoabies.

The genome sequences such as the comprehensive analysis country Pseudorabies virus epidemic strain SA, Bartha, HNQX, ZJNB, HuNXT, resist Original structure, Advance of Epidemiological Research, design is optimized to the pseudo- mad dog epitope polypeptide vaccine of restructuring.The present invention utilizes biology Hydrophily, antigenicity, plasticity, surface accessibility and the Gamier- that informatics software is carried out to its glycoprotein gB, gC, gD Robson secondary structure is analyzed, on the basis of predicting possible B cell antigen epi-position and t cell epitope, according to table Position position and the similitude of amino acid sequence, analyze each popular strain and share epitope, and with reference to the sequence letter in GenBank Breath, the epitope of prediction is compared, and further analyzes conservative of the epitope in different virus strain, so that it is determined that With 2 sections of gB Glycoprotein B cell dominant antigens epitope polypeptide, 1 section of t cell epitope, 2 sections of gC Glycoprotein Bs cell epitope, T cell table 2 sections of position, 3 sections of gD Glycoprotein Bs cell epitope.The skeleton structure of vaccine is formed after all epitopes are connected, while at skeleton nitrogen end End adds molecule adjuvant.The general structure of the 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 the coli expression carrier of embodiment two and expression bacterial strain

Designed polypeptide-coding nucleotide in embodiment one is served into the handsome biotech company's synthesis in sea, nucleotides piece Section both ends have separately designed BamH I (5 ' end) and HindIII (3 ' end) restriction enzyme site, after this 2 fragment synthesis points It is not cloned on pMD18T carriers, sequencing confirms that insertion genetic fragment is consistent with implementation sequence (see sequence table).Will restructuring Plasmid is named as pMD18T-PRV (gB/gC/gD)-BVP22, is carried out two kinds of plasmids at digestion with corresponding restriction enzyme Reason, coli expression carrier select Invitrogen companies pRSETB plasmids, also using identical restriction enzyme at Reason, digestion condition：10 μ l reaction systems, system is interior to add 2 μ l plasmids, and restriction enzyme is 5 active unit (New England biolabs), add the μ l of 10 × buffer solution 1, deionized water polishing, 37 DEG C of digestions 1.5 hours.Digestion adds after terminating Enter 1 μ l 200mM EDTA terminating reactions.In 1% agarose gel electrophoresis, electrophoresis 30 minutes.By 2.9kb under uviol lamp PRSETB plasmids, 1480bp PRV (gB/gC/gD)-BVP22 fragments are cut, and are said according to Qiagen companies gel reclaims kit Bright book carries out glue reclaim.According to carrier：The ratio of fragment 1: 2-3 individually mixes multi-epitope nucleotide fragments with expression vector, The μ l of reaction system 15, are attached by T4DNA ligases, and 16 DEG C of connections overnight, obtain recombinant plasmid and are named as pRSETB-PRV (gB/gC/gD)-BVP22 (see Fig. 1), transformed competence colibacillus e. coli bl21 (DE3) pLysS.

Conversion：PRSETB-PRV (gB/gC/gD)-BVP22 is put and melted on ice, 2 μ l Ligature liquid is added, mixes again It is even, ice-water bath 30 minutes, 42 DEG C 30 seconds, then put back to ice bath rapidly 1.5 minutes, add 1ml LB nutrient solutions, 37 DEG C, stand training Support 1 hour, 4000g low-temperature centrifugations abandon supernatant in 10 seconds, and thalline is resuspended with 200 μ lLB culture mediums；Bacterium solution is spread evenly across and contained On the LB agar plates for there are 100 μ g/mL ampicillins, culture 12-16 hours in 37 DEG C of insulating boxs are inverted in, until clone Formed.

Identification：Monoclonal on picking flat board is into LB culture mediums, 37 DEG C, 200rpm concussion and cultivates 12 hours, extracts matter Grain, double digestion is carried out using restriction endonuclease BamH I and HindIII, corresponding pseudorabies vaccines gene size fragment can be cut out Clone, 1480bp, can primarily determine that as positive colony (see Fig. 2)；Positive colony carries out determined dna sequence and is further verifying it just True property (see sequence table).

Induced expression.Positive colony is incubated overnight, morning next day by 1: 100 switching, after cultivating 3 hours, adds 0.5mM IPTG, continue culture 3 hours, prepare sample；Conventional SDS-PAGE testing goals protein expression situation --- in 56KD (see Fig. 3) at molecular weight, specific band is seen as correct clone；Correct clone, amplification culture are taken, SDS-PAGE verification tables reach After correct, further confirm that it expresses accuracy using conventional Western-blot (see Fig. 4)；By above-mentioned structure and identification journey After sequence, the foundation of original species word bank can be carried out using the positive colony selected as engineering bacteria, strain is named as pRSETB-PRV (gB/gC/gD)-BVP22/BL21 (DE3, Plys).

Fermentation, purifying and the emulsification of the engineering bacteria of embodiment three

Fermentation takes production strain, is inoculated in 2ml LB fluid nutrient mediums and (contains 100 μ g/ml ampicillins), 37 DEG C, 12 hours activated spawns of 180rpm shaken cultivations.Shaking flask, 37 DEG C of shaken cultivations to OD600=are accessed with 1: 100 inoculum concentration again 3, it can be inoculated with 10% ratio into fermentation tank.Fermentation is semisynthetic medium with culture medium, is prepared with distilled water, wherein not containing Any antibiotic.Dissolved oxygen and pH value electrode are corrected, opens tank body stirring, revolution 300rpm, tank body sterilizes online, treats to train in tank When nutrient solution temperature is down to 37.0 DEG C, pH and dissolved oxygen (OD) zero point are demarcated.Fermentation temperature is 37.0 ± 0.1 DEG C, and dissolved oxygen control exists 20% or so, pH control flow feeding 500ml when cultivating thalline OD600=1.0~1.2 after 7.0, inoculation, 1 hour after feed supplement IPTG (final concentration of 0.5mM) induced expression is added, 6 hour after fermentation of continuous induction terminate, and SDS-PAGE calibratings are done in sampling Expression.

The thalline that will be collected into is purified, it is mixed with occlusion body washing lotion I (1%Triton X-100,20mMTris-cl PH8.0) Ultrasound, 2000W ultrasonic degradations 1 hour are carried out after outstanding.4 DEG C, 12000rpm is collected by centrifugation occlusion body, and with occlusion body washing lotion II (1%DOC, 4M urea, 20mMTris-cl PH8.0) suspension twice ultrasonic washs occlusion body, and secondary low-temperature centrifugation is collected and included Body.Occlusion body precipitation 8M urea, 0.3% β-ME, 20mM Tris-cl (pH=8.00) are mixed, are stirred at room temperature 4 hours, 8000rpm low-temperature centrifugation 30min, discard precipitation.Albuminate 1: 100 dilutes, renaturation solution Tris (PH8.0) buffer system, 0.3M arginine is added, 4 DEG C are stirred renaturation 24 hours.Renaturation solution pH=8.0 20mM phosphate buffers, 0.5M sodium chloride, 20mM imidazoles, affinity column in balance, with pH=8.0 20mM phosphate buffers, 0.5M sodium chloride, the elution of 0.5M imidazoles； 1.5M (NH are used again₄)₂SO4,100mMEDTA, pH=8.5 10mM Na₂HPO₄Hydrophobic chromatography post in balance, releveling, uses pH =8.5 10mM Na₂HPO₄Elution, produce pseudo- mad dog polypeptide epitope vaccine semi-finished product stoste.It is SDS-PAGE and Western Whether blot markings calibrating purified product is target protein (Fig. 5, Fig. 6).

The semi-finished product of purifying are diluted to 200 μ g/ml by emulsification with the PBS of sterilizing.Take import white mineral oil adjuvant DUOPRIME (pharmaceutical grade) sterilizes 15 minutes through 121 DEG C, standby.In oil phase: aqueous phase=50: 50 ratio is prepared, first by oil phase Add in emulsion tank, start mixer and be slowly stirred with 80-100r/min speed, be slowly added into aqueous phase, be stirred for after adding 2min, 9min is then emulsified with 5500r/min high-speed circulatings, the single-phase vaccine of Water-In-Oil is made.

The pseudo- mad dog epitope polypeptide vaccine safety experiment of example IV

Material

Vaccine：Pseudo- mad dog epitope polypeptide vaccine, lot number 20120604,20120608,20120612.

Experimental animal (1) 18-22g small white mouses 17, purchased from medicine inspecting institute of Qingdao City.(2) in-pig 8,5 week old piglets 12, IDEXX ELISA commercial kits detection PRV is negative, Guangdong Winsun Bio-Pharmaceutical Co., Ltd.'s experimental animal center There is provided, pig environment before starting to test adapts to one week.

Method

Security of the vaccine to small white mouse

Small white mouse is subcutaneously injected, every injection 0.5ml, every batch of vaccine injection 5,15 small white mouses of co-injection, sets simultaneously 2 negative controls, Continuous Observation 10 days calmly, observe the health status of small white mouse.

Security of the vaccine to in-pig

6 female Landraces were in gestation 40~60 days, the pseudo- mad dog epitope polypeptide vaccine of intramuscular injection lmL respectively, per batch Immune two, 2 sows are set in addition and are used as non-immunized controls, observe farrowing situation.

Security of the vaccine to piglet

9 epitope polypeptide vaccines provided for injection center of PRV negative antibodies piglet, 3 pigs of every batch of vaccine injection, Every pig posterior auricular muscle meat injects 1ml and amounts to 2ml.Negative control 3, every injection white-oil adjuvant 2ml are set up simultaneously, and clinic is seen Examine 14 days.Off-test, every pig are weighed.

As a result

Safety testing of the vaccine to small white mouse

As a result such as the slightly rise of the table animal subject second day of 1,20120612 immune group 1 body temperature, recover within the 3rd day normal, Appetite and health status are without exception, consistent with control group, no dead to occur, it is seen that pseudo- mad dog epitope polypeptide vaccine is to small white mouse Safety, it is shown in Table 1.

Safety testing result of the vaccine of table 1 to small white mouse

Group	Size of animal	Body temperature	Appetite	Health status	The dead quantity
						20120604	5	1 body temperature rise, other are normal.	Normally	It is in a good state of health	0
20120608	5	Normally	Normally	It is in a good state of health	0
						20120612	5	Normally	Normally	It is in a good state of health	0
Control	2	Normally	Normally	It is in a good state of health	0

Safety testing of the vaccine to in-pig

Show that testing sow raises to childbirth by the result of table 2, except having other a weak tire and stillborn foetus, do not occur miscarriage, Premature labor and production mummy tire phenomenon, it was demonstrated that this vaccine is safe and reliable to in-pig, to the production performance of in-pig Do not influence.Vaccine group and control group test pig do not occur abnormal clinical reaction during experiment, and spirit, feeding are normal, note Penetrating position does not have inflammatory symptom.

The farrowing sow production performance result of table 2

Safety testing of the vaccine to piglet

As a result as table 3, vaccine group and control group test pig do not occur abnormal clinical reaction, spirit, normal, note of searching for food Penetrating position does not have inflammatory symptom, and body temperature is normal, and this vaccine group of immune group piglet average weight gain is respectively 2.5Kg, 2.4Kg, empty White control group average weight gain 2.3Kg, significant difference (P ＞ 0.05) is not presented between group.Refer to table 1.Whole 14 days experimental observation phases Interior, all immune totally 9 first 5 week old piglets, body temperature and appetite are normal, any clinical anomaly, experiment knot do not occur Shu Hou, 9 piglets are strong to live, with control group indifference.This explanation, pseudo- mad dog epitope polypeptide vaccine is safe to piglet.

The piglet safety detection result of table 3

The animal packet of embodiment five is with being immunized

For the efficiency evaluation parameter as much as possible for obtaining vaccine, after the present invention have detected immune balb/c mice Specific antibody, mouse and the virucidin of piglet and the relevant T lymphopoiesis situations of cellular immunity.

1 vaccine is with attacking poison virus

Pseudo- mad dog epitope polypeptide vaccine is provided by Hongqiao Ming Qin research and development centres, lot number 20120604,20120608, 20120612, gE gene nature deletion of vaccine strain (Bartha-K61) live vaccines are given by Guangdong Yongshun pharmaceutical development center, Lot number is 2012016.

2 experimental animals and packet

The BalB/C female mices of 25 5 week old, 5 groups are randomly divided into, three lot number groups of epitope polypeptide vaccine, live vaccine group And control group, every group of 5/cage；25 first 5~6 week old piglets, male and female half and half, it is divided into 5 groups immediately, epitope polypeptide vaccine three Lot number group, live vaccine group and control group, every group 5, experimental animal is raised one week in advance before experiment, adapts to environment.

3 experimental designs and method

Balb/C mouse and piglet are immunized according to packet situation, once, 20 μ g/ are only for two weeks booster immunizations after head exempts from Or 20 μ g/ heads.Head exempt from after 2 weeks, 4 weeks, 6 weeks, 8 weeks, mouse docking blood sampling simultaneously separates serum, and after blood sampling for the last time, government official Mouse is killed, separation hamster kidney cell is used for T lymphocyte proliferation assays；Head exempts from latter 6 weeks, 8 weeks, 10 weeks, piglet vena cava anterior blood sampling And separate serum and PBMC and detected for cellular immunity.

The indirect ELISA of embodiment six detects gC antibody responses

Recombinant protein purification sample is diluted to 1 μ g/ml with 50mmol/l CBS (PH9.6), adds 96 hole elisa plates, often The μ l of hole 100,4 DEG C are coated with overnight.PBST washing lotions buffer washing three times after, with the horse serum of confining liquid 5% (PBS buffer systems) in 37 DEG C, close 1 hour.After washing three times, mouse serum sample is diluted to confining liquid (5% horse serum, PBS buffer systems) 1: 20,100 μ l are added per hole, two repetitions, 37 DEG C are incubated 1 hour；After washing three times, added per hole and be diluted to 1: 10000 HRP- mark secondary antibodies (sheep anti mouse), 37 DEG C are incubated 1 hour；Washing three times after, per hole add 50 μ l tmb substrates, room temperature, lucifuge, 10min is reacted, adds 2M H₂SO₄Stop reaction.Light absorption value (ELIASAs of BIORAD 680) is read under 450nm wavelength.

As a result

As shown in fig. 7, different groups produce the gC protein specific antibodies of varying level.During whole experiment, epitope polypeptide Vaccine and live seedling group mouse generate obvious gC protein-specifics IgG antibody, are in pole significant difference (P ＜ with compareing 0.01).As a result show that epitope polypeptide vaccine provided by the invention can stimulate Balb/C mouse to produce humoral immune reaction, and it is anti- Body is horizontal suitable with existing live vaccine, no significant difference (P ＞ 0.05).

The antibody neutralization test of embodiment seven

Exempt from the serum of collection in rear 4,6,8 weeks to head, carry out virucidin (SN) detection.Blood sample in serum is carried out and 56 DEG C of inactivation 30min are carried out before experiment, the 100TCID of equivalent is added after being serially diluted for twice₅₀PRVBartha-K61 strains, add 96 well culture plates, 5%CO₂Incubator (Sanyo MCO-15AC), 37 DEG C are cultivated 1 hour, then, add 100 μ l Vero cells Suspension, contain 2 × 10 per hole⁴Individual cell.Culture plate is placed in 5%CO₂Moist environment, 37 DEG C cultivate 3 days, read survey viral dilution The cytopathy of degree, each number for infecting hole are expressed as the ratio that every viral dilution is inoculated with each hole count.Dilution factor is not to see The highest extension rate for observing CPE is defined.Each sample does three repetitions.

As a result

It is shown in Table after 4, head exempts from 4 weeks (2 weeks booster immunizations after head exempts from) and detects neutralizing antibody first in all immune groups.It is whole During individual experiment, neutralizing antibody caused by epitope polypeptide vaccine group and Attenuate vaccine group is on close level, but immune group is apparently higher than right According to the neutralizing antibody of group, pole significant difference (P ＜ 0.01) is presented；8 weeks after exempting to head, control group does not produce any neutralizing antibody.

The detection of mouse neutralizing antibody is immunized in table 4

Note：1 antibody titer ＜ 1: 8 is considered as no neutralizing antibody；2NR represents antibody titer without definite result；3a represents mouse Quantity

The lymphopoiesis of embodiment eight detects

In order to further contrast cell-mediated immune reaction, carried out within 8 weeks after head exempts from cell proliferation test, head exempt from after 6, 8th, Swine PBMC proliferation test has been carried out within 10 weeks, to detect the cell immune response of vaccine immunity.By hamster kidney cell or piglet PBMC It is put into 96 porocyte plates, 100 μ l/ holes (2 × 10⁵Cells/well).100 μ l/ culture mediums are then added, or are added through ultraviolet photograph The PRV of inactivation is penetrated, is mixed.Concanavalin (5 μ g/ml, Sigma) is used as positive control.Nephrocyte or PBMC samples do three Individual repetition.The method provided according to Bounous, carry out hamster kidney cell with improved MTT colorimetric methods or Swine PBMC proliferation activity is examined Survey：Hamster kidney cell 72 hours, or PBMC cultivate 96 hours after, per hole add 10 μ l WST, be incubated 5 hours, in 490nm after incubation Reading under wavelength.Stimulus index (SI) (does not stimulate) average value in hole to count with the average value in antigen stimulated cells hole than cell Calculate.

As a result

Similar with neutralizing antibody reaction, the nephrocyte that highest level is observed in epitope polypeptide vaccine group and live vaccine group increases Activity is grown, sees Fig. 8, Fig. 9.The T lymphopoiesis of control group is significantly lower than other immune groups (P ＜ 0.05), epitope polypeptide epidemic disease The stimulus index of seedling group and live vaccine group is close, and significant difference (P ＞ 0.05) is not presented.It is similar with hamster kidney cell, 6 after being immunized Week, 8 weeks, 10 weeks, to have also discovered this rule (Fig. 9) in Swine PBMC propagation detection.These as shown by data epitope polypeptide vaccines Cell immune response can be strengthened in natural host Immune inducing in vivo.

The detection of the vaccine immunity piglet cell immune response of embodiment nine

Method is shown in embodiment seven, the results are shown in Table 5.Head exempt from after 6 weeks, 8 weeks, 10 weeks, collection blood serum sample is for viral neutralization Experiment.During whole experiment, control group piglet sample is not detected by PRV neutralizing antibodies, and this is consistent with the result of hamster kidney cell.Institute There is immune group to detect neutralizing antibody within 6 weeks after head exempts from, latter 8 weeks, 10 weeks epitope polypeptide vaccine groups are immunized and live vaccine group is whole Immune piglet detects neutralizing antibody.Epitope polypeptide vaccine group has four-head piglet neutralizing antibody titers to reach 1: 32, living virus vaccine group There is one to reach 1: 32；For live vaccine group 10 weeks after immune, it is 1: 8 to have a pig neutralizing antibody titers, and other immune piglets are equal Reach 1: 16.

The detection of piglet neutralizing antibody is immunized in table 5

Note：1 antibody titer ＜ 1: 8 is considered as no neutralizing antibody；2NR is represented without definite result；3a represents the quantity of pig.

Claims

1. a kind of bursa of farbricius protein engineering vaccine fusion protein, its amino acid sequence is SEQ ID No.2.

2. a kind of nucleic acid molecules, it encodes claim 1 fusion protein.

3. a kind of carrier, it contains the nucleic acid molecules described in claim 2.

4. a kind of host cell, it contains the carrier described in claim 3.

5. the nucleic acid molecules described in claim 2, its particular sequence is as follows：

6. a kind of vaccine for being used to prevent pseudoabies, it includes the fusion protein described in claim 1 and can pharmaceutically connect The carrier received.

7. application of the fusion protein in pseudo- mad dog epitope polypeptide recombinant vaccine is prepared described in claim 1.