CN104628871B - A kind of preparation for recombinating bursal disease protein engineering vaccine - Google Patents

Abstract

The present invention relates to a kind of preparation and application of the restructuring bursa of farbricius (Infectious Bursal Disease, IBD) protein engineering vaccine.The vaccine is used as vaccine frame structure using IBDV major structural proteins VP2, VP3 B cell neutralizing epitope and T cell immune epitope, by being connected again with cell factor fowl interleukin 15 (chIL 15) after flexible linker connections, Escherichia coli are converted after being cloned into pRSETB carriers, through techniques such as everfermentation, purifying, emulsifications, the bursa of farbricius protein engineering vaccine with Desirable immunogenic is obtained.The invention further relates to the application method of the vaccine.Zoopery shows; it is suitable with living virus vaccine in humoral immunity and effect on cellular immune level to recombinate bursa of farbricius protein engineering vaccine; it can on a cellular level stimulate and produce T lymphopoiesis immune responses; produce the antibody mediated immunity with viral neutralization activity on body fluid levels to react, protest test shows that recombinating bursa of farbricius protein engineering vaccine group positive effect is better than live vaccine group.

Description

A kind of preparation for recombinating bursal disease protein engineering vaccine

Technical field

The invention belongs to biotechnology genetic engineering field, relates generally to a kind of restructuring bursal disease protein engineering vaccine system It is standby with application.Specifically, using gene recombination technology, by major structural protein VP2, VP3 epitope and cell factor fowl IL-15 Series connection, and carrier is cloned into, Host Strains are converted, through everfermentation, prepared by purifying, emulsifying process, obtains recombinating bursa of farbricius albumen work The application of engineered vaccine and the vaccine in avian infectious disease bursal disease viral disease is prevented.

Background technology

Bursal Disease (Infeetious Bursal Disease, IBD) is by infectious bursa of Fabricius virus A kind of acute, highly contagious disease caused by IBDV, it is to endanger one of poultry husbandry infectious disease the most serious at present (Ikuta et al., 2001；Van den Berg, 2000).This virus is without cyst membrane, in icosahedron cubic symmetry.There is individual layer Capsid, nucleocapsid are made up of 32 a diameter of 12nm capsomere, diameter about 60nm.It is also common free in addition to complete virus particle Capsid, in infection cell, virus is often in lattice-like arrangement.

IBDV genomes are made up of two AMPLIGEN sections, respectively A sections (big section) and B sections (small section) (Lombardo et al., 2000), two sections include 5 ' end noncoding regions (5 ' non-coding region, 5 '- NCR), code area and 3 ' end noncoding regions (3 '-NCR).IBDV genome A, B sections encode 5 kinds of albumen altogether.B sections encode VP1 Albumen (95kDa), it is referred to as VPg (Delmas et al., 2011, Muller et as genome connection albumen in virion Al., 2003).A sections include two partly overlapping ORF, small ORF coding VP5 albumen (17kDa), and big ORF encodes one point Son amount for 108kDa polyprotein, the albumen by self cleavage produce VP2 precursor proteins pVP2 (48kDa), VP3 (32kDa), VP4 (28kDa), pVP2 are further processed into as the VP2 (40kDa) of maturation by c-terminus.VP2 and VP3 is structural proteins, structure Into the ectonexine of virion capsid, containing neutralizing epitope (Azad et al., 1991；Heine and Boyle, 1993； Villegas et al., 2008；Yip et al., 2007).

VP2 by IBDV A sections VP2 gene codes, be IBDV major structural protein, account for viral total protein 51%, form the outer capsid of virus.VP2 and another major structural protein VP3- acts the skeleton for forming IBDV nucleocapsids.VP2 is carried IBDV depends on the protectiveness neutralizing antigenic site of protein tridimensional three-dimensional conformation, can induce and produce protectiveness neutralizing antibody, And neutralizing antibody with serological specificity its induction passively protect host do not infected by IBDV (Lukert and Saif, 2003).VP2 contains conformation dependent (discontinuous) antigenic determinant that can induce generation neutralizing antibody of serotype specificity, Its neutralizing antibody induced can protect host not infected by IBDV, therefore VP2 is IBDV host protective antigen (Azadetal., 1991.Therefore, it is considered that VP2 is IBDV host protective antigen, VP2 amino acid sequences between different strains The difference of row, which is mainly concentrated, is present in the 206th~350 totally 145 amino acids residue, referred to as VP2 hypervariable regions, and the region is lucky Between two restriction enzyme sites AccI and Spel (Rautenschlein et al., 2005).Containing two hydrophilic peak A (the 210th~225) and peak B (the 312nd~324) and between region, seven amino behind the second hydrophilic area The heptapeptide area of acid composition, commonly referred to as this region is VP2 hypervariable regions, belongs to neutralizing epitope, can identify weak malicious and strong poison.First parent Pool contributes to the stabilization of polypeptide conformation, and identification of second hydrophilic area to viral neutralizing monoclonal antibody is relevant.Neutralize single The epitope point that clonal antibody is identified is a three-dimensional three-dimensional conformation (Coulibaly et al., 2005).It can draw Playing the amino acid variation of epitope point three-dimensional conformation generation minor variations all can make IBDV antigenicity change (Letzel et al., 2007, Martinez-Torrecuadrada et al., 2003).Different strains are in hypervariable region Make a variation larger, the change of molecular structure can cause the change of the change and host of virus virulence to vaccine response so that tradition Vaccine strain can not control that it is popular (Pitcovski et al., 2003；Rong et al., 2007).In addition, VP2 is cell Cause after infection IBDV one of Apoptosis principal element (Ikuta et al., 2001；Van den Berg, 2000).

VP3 albumen is one of main structural proteins of IBDV, and molecular mass is about 32kD, accounts for the 40% of viral total amount, position In the inner surface of virion, played an important role during IBDV nucleocapsids skeleton is formed.VP3 contains group specific antigen Determinant, the generation of neutralizing antibody is can induce, but respond is extremely weak, and immune protective is not strong.VP3 is for virus-like particle Formation is necessary, and formation and form of the VP3 for viral capsid are completely required (Lee et al., 2003).VP3 is extremely The non-overlapped non-neutral epitope of two independences less be present, one of them is that another is common to two serotypes Serotype special (Martinez-Torrecuadrada et al., 2003).Different blood serum subtype IBDV VP3 gene phases To conservative, VP3 presence is the premise that virus-like particle (VLPs) can be properly formed, and it is formation and the shape of IBDV capsids State be completely it is required, its effect is to stablize viral RNA.In addition, because the how individual sections of VP3 have hydrophily, make it in disease Poison is with playing facilitation (Lee et al., 2003) during antibody response.

Interleukin 15 (interleukin 15, IL.15) is a kind of newly discovered cell factor, IL-15 many property Matter is considered as same IL-2 similar with function, as inducing T cell proliferation, improve NK (NK) cytotoxicity, Promote (Grabstein K H, 1994) such as B cell proliferation and the differentiation of activation.Lillehoj et al. is in cell cdna library A new gene is cloned into, its 5 ' end noncoding region contains two outer frame ATG initiation codons, and encoding proteins contain longer Signal peptide (is made up of) 66 amino acid, and maturation protein contains four highly conserved cysteine residues.The albumen is in vitro The growth for the chicken T lymphocytes that ConA activates can be maintained and strengthen the killing activity of NK cells, this and the mammal reported IL-15 gene is similar to protein specificity, thereby determines that gene code ChIL-15 (Lillehoj H S etal, 2001). IL-15 is a kind of multi-functional cell factor, includes (1) maintenance T cell strain CTL-2 growth, and inducing cytotoxic T cell is lived Property, activation, the differentiation of pre-sensitization T cell are induced, promotes intestinal mucosal lymphocytes particularly TCR cell differentiations to grow, Er Qieyu IL-2 collaborations low concentration can cause T cell rapid propagation (Chu C L etal, 1999；Schluns K S etal, 2002) (2) to normal B cells, after with PMA and anti-IgM pre-activate, IL-15 can induce its breeder reaction, when by IL- 15 with CD40L use in conjunction when, B cell IgG secretion (Armitage R T, 1995) can be made；(3) can be with point of induced NK cell Be melted into ripe, strengthen its killing activity, can also significantly enhance NK cells expression IF γ and TNF α (Wei X Q, 2001；Waldmann T A, 2001)；(4) normal peripheral blood mononuclear cells PBMC can be stimulated to breed；(5) IL-15 of high concentration can increase LPS activation Mononuclear macrophage produce proinflammatory inflammation factor such as IL-1, IL-6 and TNF α, the IL-15 of low concentration can selectivity suppression it is proinflammatory The generation of inflammation factor, confrontation inflammatory factor is without influence.(6) suppress lymphocyte and apoptosis occurs, and the thin of IL-2 mediations can be suppressed Born of the same parents are dead (Waldmann T A, 2001).

Traditional inactivation and living virus vaccine inoculation are all once used as prevention and control IBDV Main Means.However, inactivated vaccine lacks induction machine Body produces the ability of solid immunity, especially in terms of cell-mediated immune efficacy.Threatened in face of vvlBDV, inactivated vaccine Chicken can not be made to be protected within whole growth period., may be right after being immunized because attenuated IBDVs vaccine is not often attenuated completely The lymph follicle of the bursa of farbricius causes certain pathology damage, or even what is had can also cause immunosupress (Rautenschlein Et al., 2005), can also cause the appearance of high virulence or variant (Snyder, 1990).Moreover, some IBDV variants are lived Vaccine cause immunosupress and subclinical scabies secondary infection (Fussell, 1998；Donnelly et al., 2000).Therefore, develop A kind of novel I BDV vaccines of safely and effectively suitable mass immunization are imperative.

Summary of the invention

The present invention is made with major structural protein VP3B cell epitopes and VP2B cells Neutralization and crystallization and t cell epitope For vaccine frame structure, with avian cytokines interleukin 15 (chIL-15) gene tandem, after expression in escherichia coli, pass through The techniques such as protein purification, emulsification, obtain the recombinant vaccine with Desirable immunogenic.Can induce after this vaccine immunity target animals has The humoral immunity and cell immune response of effect.

An object of the present invention is the provision of a kind of new recombinant protein engineering that can be used to prevent bursa of Fabricius in poultry Vaccine polypeptide and its vaccine combination；The second object of the present invention is the provision of the structure of the bursa of farbricius protein engineering vaccine And preparation method；The third object of the present invention is the provision of the genetic engineering that can express the bursa of farbricius protein engineering vaccine Bacterial strain；The fourth object of the present invention is the provision of the preparation method of the protein engineering vaccine；The fifth object of the present invention exists In providing purposes of the bursa of farbricius protein engineering vaccine in bursal disease is prevented.

In a first aspect, the invention provides a kind of restructuring bursa of farbricius protein engineering vaccine for being used to prevent bursal disease is more Peptide and combinations thereof.It is thin that it contains major structural protein VP3 protein Bs cell epitope, VP2 protein B cell Neutralization and crystallizations and T Born of the same parents' epitope and avian cytokines interleukin 15 (chIL-15).Described bursa of farbricius protein engineering vaccine protein or polypeptide or pharmacy Carrier required for upper acceptable salt and expression epitope protein.Carrier can also include separately encoded each epitope Sequence, series connection can be carried out by genetic engineering method.The vaccine also includes nonimmune active material, as each polypeptide Coupling part, the immunogenicity without epitope, also without any adjuvanticity, mainly have purification tag, joint peptide, Chemical modification part, N-terminal signal peptide and C-terminal polyadenylic acid etc..The pharmaceutically acceptable salt refer to it is non-toxic, stimulate and Allergy, suitable for the salt of human or animal tissues.Inert matter and pharmaceutically acceptable salt are those skilled in the art It is known.It is as follows to recombinate bursa of farbricius protein engineering vaccine polypeptid acid sequence：

LGMAQPTQNSAGARRRPESQKTHVKSICLQYQLYLLLNSYFFCLLKNKTGLTIFFLCAYVPKTEANHCK WSDVLKDLELIKTSEDIDVSLYTANTYEDIECQEPVMRCFFLEMKVILHECDIKKCSRKHDVRNIWKNGNARFATYQ LNSTTAKKCKECEEYEEKNFTEFIQSFVKVIQRECRKYANGDIPGCKTNLQDQTQGGTTGPASIPDDTLEKHTLRSE TSTGSGLQSNGNYKFGGVATCDSSDRPRIYTIAAANDYQFSSQYQADGGSGPNPELAKNLITEYGRFDPGAMNYTKL ILSERDRLGIKTVWPTREYTDFREYFMEGGPSCDPNAHRMRNFLANAPQAGSKSQRAKYGTAGYGVEARGPTPEEAQ REKDTRISKKMEGSGNGHRGPSPGQLKYWQNTREIPDPNEDYLDYVHAEKSRLASEEQIGSGNHGRGPNQEQMKDLL LTAMEMKHRNPRRAPPKPKPKPNAPTQRPPGRLGR

In second aspect, the invention provides a kind of nucleic acid molecule, and it encodes the Fa Shi described in first aspect present invention Capsule protein engineering vaccine polypeptide.Nucleotide of the present invention can be rna form, DNA form, be synthesized by artificial synthesized mode more Epitope tandem sequence and cytokine sequence, then operate connection rear clone by genetic engineering and enter carrier, be transformed into big Enterobacteria, screening, fermentation, bursa of farbricius protein engineering vaccine polypeptide is obtained after purification.The nucleic acid can be carried out in the present invention Conventional molecular biology manipulations, 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 preferred that the nucleotide sequence in the present invention is as follows：

CTG GGG ATG GCA CAG CCA ACA CAA AAC TCT GCC GGA GCA CGG AGAAGG CCG GAG AGT CAG AAA ACA CAT GTG AAA AGT ATT TGT CTC CAG TACCAA CTG TAT CTA CTT TTG AAC AGC TAT TTC TTT TGC CTT TTA AAG AAT AAGACT GGA CTA ACC ATC TTC TTC CTA TGT GCT TAT GTA CCA AAG ACA GAAGCA AAT CAC TGT AAG TGG TCA GAC GTT CTG AAA GAT TTG GAG CTG ATCAAG ACA TCT GAA GACATT GAC GTCAGT TTA TAT ACT GCA AAC ACA TAC GAG GAT ATA GAA TGC CAG GAA CCT GTA ATG AGA TGT TTT TTT TTA GAGATG AAA GTG ATT CTT CAC GAA TGT GAT ATC AAA AAA TGT AGT AGG AAGCAT GAT GTA CGG AAC ATA TGG AAA AAT GGA AAT GCA AGA TTT GCA ACTTAC CAG TTG AAT TCC ACA ACA GCA AAA AAA TGC AAA GAA TGT GAA GAGTAT GAA GAAAAAAAT TTT ACA GAA TTT ATA CAG AGT TTT GTA AAG GTTATA CAG AGG GAA TGC AGA AAA TAC GCT AAC GGT GAT ATC CCA GGT TGCAAG ACA AAC CTG CAA GAT CAA ACC CAA GGT GGT ACA ACC GGA CCG GCGTCC ATT CCG GAC GAC ACC CTA GAG AAG CAC ACT CTC AGG TCA GAG ACCTCG ACC GGT TCT GGT CTG CAG AGC AAT GGG AAC TAC AAG TTC GGT GGTGTA GCA ACA TGT GAC AGC AGT GAC AGG CCC AGA ATC TAC ACC ATA GCTGCA GCC AAT GAT TAC CAA TTC TCA TCA CAG TAC CAA GCA GAT GGA GGTTCT GGT CCA AAT CCT GAA CTA GCA AAG AAC CTG ATC ACA GAA TAC GGCCGA TTT GAC CCA GGA GCC ATG AAC TAC ACA AAA TTG ATA CTG AGT GAGAGG GAC CGT CTT GGT ATC AAG ACC GTA TGG CCA ACA AGG GAG TAC ACTGAC TTT CGC GAG TAC TTC ATG GAG GGT GGT CCA TCT TGT GAC CCC AACGCC CAT CGG ATG CGT AAT TTT CTC GCA AAC GCA CCA CAG GCA GGC AGCAAG TCG CAA AGA GCC AAG TAC GGG ACA GCA GGC TAC GGA GTG GAG GCCCGA GGC CCC ACA CCA GAG GAG GCA CAG AGG GAA AAA GAC ACA CGG ATCTCA AAG AAG ATG GAG GGT TCT GGT AAT GGG CAC AGG GGG CCT AGC CCCGGC CAG CTA AAG TAC TGG CAG AAC ACA CGA GAA ATA CCT GAT CCA AACGAG GAC TAC CTA GAC TAC GTG CAT GCA GAG AAG AGC CGG TTG GCA TCAGAA GAA CAA ATC GGT TCT GGT AAC CAT GGG CGT GGC CCC AAC CAA GAACAG ATG AAA GAT CTG CTC TTG ACT GCG ATG GAG ATG AAG CAT CGC AATCCC AGG CGG GCT CCA CCA AAG CCC AAG CCA AAA CCC AAT GCT CCA ACACAGAGACCC CCT GGT CGG CTG GGC CGC

In the third aspect, the invention provides a kind of carrier, and it is except containing the compiling method described in second aspect of the present invention Family name's capsule protein engineering vaccine nucleotide acid molecule, also contain with the exercisable connection of the nucleotide sequence, procaryotic cell expression (turn Record and translation) needed for expression control element.Most basic expression control element includes promoter, transcription terminator, enhancer, Selected marker etc., these controlling elements are known in the art.Preferred e. coli bl21 (DE3, Plys) is made in the present invention For expression vector.

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 bursa of farbricius protein engineering vaccine polypeptide needed for fermentation expression production.

At the 5th aspect, the invention provides a kind of preparation method of bursa of farbricius protein engineering vaccine, it includes following step Suddenly：Engineering bacterium fermentation expresses bursa of fabricius vaccine polypeptide, by thick purifying and polishing purification technique and follow-up emulsifying process, obtains Required polypeptide.The method being directed to includes but is not limited to bacterial cell disruption, inclusion body washing, centrifugation, denaturation, affine layer Analysis, hydrophobic chromatography, anion-exchange chromatography, reverse-phase chromatography, renaturation, emulsification etc..The preparation method being related in the present invention is ability Known to field technique personnel.

At the 6th aspect, the invention provides a kind of restructuring bursa of farbricius protein engineering epidemic disease for being used to prevent bursa of Fabricius in poultry Seedling, it includes the polypeptide and pharmaceutically acceptable carrier described in first aspect present invention.The bursa of farbricius protein engineering epidemic disease Seedling can prevent the outburst of bursa of Fabricius in poultry.Pharmaceutically acceptable carrier of the present invention is immunopotentiator or immune assistant Agent, preferably immunologic adjuvant are import white-oil adjuvant.

At the 7th aspect, the invention provides the application of the restructuring bursa of farbricius protein engineering vaccine described in the 6th aspect.Epidemic disease Seedling can necessarily effective dose intramuscular injection, intracutaneous or inoculated with subcutaneous injections animal, the antibody and cell of sufficient amount can be produced The factor (such as IFN), there is provided antiviral activity, attack of the protection animal from bursal disease virus prevalence strain.In addition, in this hair In bright embodiment, by carrying out target animals attack malicious contrast test, laboratory safety is tested etc. to vaccine, show the present invention Described restructuring bursa of farbricius protein engineering vaccine is safe (see example IV), and animal can be protected from bursal disease virus sense Dye is (see embodiment five, six, seven, eight, nine, ten).

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 restructuring bursa of farbricius protein engineering vaccine expression vector pRSETB-IBDV (VP2/3)-chIL15 structure figures； Fig. 2 pRSETA-PRRSV (GP5/6/7/Ub) vector plasmid cleavage map, wherein swimming lane 1 are DNAmarker, from top to bottom molecular weight 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp are followed successively by, swimming lane 2 is empty plasmid, and swimming lane 3 is plasmid enzyme restriction figure, Swimming lane 4 compares for non-digestion；Fig. 3 SDS-PAGE detection figures, wherein swimming lane 1 is albumen Marker, is followed successively by from top to bottom 97.2KD, 66.4KD, 44.3KD, 29.0KD, 20.1KD, 14.3KD, swimming lane 2 are non-induced samples, and swimming lane 3 is induced samples, The signified destination protein for expression of arrow；Fig. 4 Westernblot detection figures, wherein swimming lane 1 is pre-dyed marker, from top to bottom 97KD, 66KD, 43KD, 31KD, 20KD, 14KD are followed successively by, swimming lane 2 is purpose albumen.Fig. 5 schemes for fermented sample SDSPAGE：Its Middle swimming lane 1 is albumen Marker, is followed successively by 97.2KD, 66.4KD, 44.3KD, 29.0KD, 20.1KD, 14.3KD from top to bottom, Swimming lane 2 is non-induced samples, and swimming lane 3 is induced samples, and signified arrow is the destination protein expressed；Fig. 6 is restructuring vaccine protein The more anti-reaction result figure with wild virus infection serum, wherein swimming lane 1 is pre-dyed albumen marker, be followed successively by from top to bottom 116KD, 66KD, 45KD, 35KD, 25KD, 20KD, 10KD, swimming lane 2 are wild poison and recombinant vaccine antiserum westernblot results, swimming lane 3 be restructuring VP2/3-IL15 albumen and wild malicious antiserum westernblot results；Fig. 7 detects for target animals IgY antibody titers； Fig. 8 is the association reaction result of immune antiserum and IBDV vaccine strains；Fig. 9 is recombinant protein VP2/3-IL15 and live vaccine K85 The sero-fast association reaction result of strain；Figure 10 recombinant VP 2s/3-IL15 proteantigens stimulate T cell breeder reaction；Figure 11 K85 epidemic diseases Seedling poison antigenic stimulus T cell breeder reaction.

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 bursa of farbricius protein engineering vaccine protein of embodiment one

The present invention utilizes correlation according to current domestic bursa of farbricius Major Epidemic strain Viral structural protein VP2, VP3 amino acid sequences The strain of bioinformatics software DNASTAR, BIMAS and SYFPEITHI pop carries out B cell neutralizing epitope and t cell epitope point Analysis, while introduce avian cytokines interleukin 15 (IL-15) and be used as adjuvant molecules.Designed bursal disease virus B cell and T is thin Co-expressed after born of the same parents' epitope and the series connection of interleukin 15 molecular polypeptide in Escherichia coli, through techniques such as everfermentation, purifying, emulsifications, Obtain the bursa of farbricius protein engineering vaccine with Desirable immunogenic.The vaccine prepared using the present invention can effective Yu Fangfashi Capsule disease.

Comprehensive analysis country bursa of farbricius viral prevalence pnca gene group sequence, antigenic structure, Advance of Epidemiological Research, counterweight Design is optimized in group bursa of farbricius protein engineering vaccine.What the present invention was carried out using bioinformatics software to its structural proteins Hydrophily, antigenicity, plasticity, surface accessibility and Garnier-Robson secondary structure are analyzed, and predict possible B On the basis of cell antigen epitope and t cell epitope, according to the similitude of epitope position and amino acid sequence, each prevalence is analyzed Strain shares epitope, and with reference to the sequence information in GenBank, the epitope of prediction is compared, further divided Analyse conservative of the epitope in different virus strain, so that it is determined that with VP2 structural proteins T cell dominant antigens epitope polypeptide 1 Section, 2 sections of B cell epitope, 3 sections of VP3 structural proteins B cells epitope.The skeleton structure of vaccine is formed after all epitopes are connected, together When skeleton nitrogen end add molecule adjuvant.The general structure of the vaccine is：

Molecular Adjuvant(chIL15)-VP2T Cell Epitope-VP2B Cell Epitope 1-VP2B Cell Epitope 1-VP2 B Cell Epitope 2-VP3B Cell Epitope 1-VP3B Cell Epitope 2- VP3B Cell Epitope 3

The structure of the coli expression carrier of embodiment two and expression bacterial strain

Designed polypeptide-coding nucleotide is served into the handsome biotech company's synthesis in sea, nucleotide fragments both ends difference BamH I (5 ' end) and HindIII (3 ' end) restriction enzyme site are devised, is cloned into respectively after this fragment is synthesized On pMD18T carriers, sequencing confirms that insertion genetic fragment is consistent with implementation sequence (see sequence table).Recombinant plasmid is distinguished It is named as pMD18T-IBDV (VP2/3)-chIL15.Two kinds of plasmids are subjected to digestion processing with corresponding restriction enzyme, greatly Enterobacteria expression vector selects the pRSETB plasmids of Invitrogen companies, also using identical restriction enzyme ferment treatment, enzyme Tangent 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), the μ l of 10 × buffer solution 1, deionized water polishing, 37 DEG C of digestions 1.5 hours are added.Digestion adds 1 μ l after terminating 200mM EDTA terminating reactions.In 1% agarose gel electrophoresis, electrophoresis 30 minutes.By 2.86kb pRSETB matter under uviol lamp Grain and 1467bp IBDV (VP2/3)-IL15 fragments are cut, and glue is carried out according to Qiagen companies gel reclaims kit specification Recovery.According to carrier：Fragment 1：2-3 ratio individually mixes multi-epitope nucleotide fragments with expression vector, the μ of reaction system 15 L, it is attached by T4DNA ligases, 16 DEG C of connections overnight, obtain recombinant plasmid and are respectively designated as pRSETB-IBDV (VP2/ 3)-chIL15, (see Fig. 1), transformed competence colibacillus e. coli bl21 (DE3) pLysS.

Conversion：PRSETB-IBDV (VP2/3)-chIL15 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 respectively, corresponding bursa of fabricius vaccine gene size fragment can be cut out Clone, 1467bp, can primarily determine that as positive colony (see Fig. 2)；Positive colony carries out determined dna sequence and further verifies it Correctness (see sequence table).

Induced expression.Positive colony is incubated overnight, morning next day by 1: 100 switching, after cultivating 3 hours, adds 0.2mM IPTG, continue culture 4 hours, prepare sample；Conventional SDS-PAGE testing goals protein expression situation --- in 56KD (see Fig. 3), specific band is seen as correct clone；Correct clone is taken, amplification is cultivated, after SDS-PAGE verification tables reach correctly, Further confirm that it expresses accuracy using conventional Western-blot (see Fig. 4)；, can after above-mentioned structure and evaluation program Foundation using the positive colony selected as engineering bacteria progress original species word bank, strain name pRSETB-IBDV (VP2/3)- IL15/BL21 (DE3, Plys).

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

Fermentation takes production strain, is inoculated in 2m1LB fluid nutrient mediums and (contains 100 μ g/ml ampicillins), 37 DEG C, 12 hours activated spawns of 200rpm 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 40% 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 0.2mM) induced expression is added, 5 hour after fermentation of continuous induction terminate, and SDS-PAGE calibratings are done in sampling Expression (see Fig. 5).

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； Produce restructuring bursa of farbricius protein engineering vaccine semi-finished product stoste.

The semi-finished product of purifying are diluted to 100 μ g/ml by emulsification with the PBS of sterilizing.Follow the example of Guo Sai BIC Corps Montanide ISA 50V2 adjuvants, by 121 DEG C, sterilize 15 minutes, it is standby.In oil phase: aqueous phase=50: 50 ratio is matched somebody with somebody System, first oil phase is added in emulsion tank, mixer is started and is slowly stirred with 80-100r/min speed, be slowly added into aqueous phase, add 2min is stirred for after complete, 9min is then emulsified with 5500r/min high-speed circulatings, the single-phase vaccine of Water-In-Oil is made.

Example IV restructuring bursa of farbricius protein engineering vaccine safety experiment

The plumage of 1 experimental animal, 30 age in days SPF chickens 20.

2 vaccines are provided by research and development centre of company, lot number 131024,131027,131031.

20 30 age in days experimental animals are randomly divided into 4 by experiment at random using the experimental design of single-factor completely random Group, 3 lot number groups and control group, every group 5, without weight differences between group.Immune group is in every chicken leg portion intramuscular injection vaccine 0.3ml/ heads, observe to 10 days, control group uses physiological saline emulsion 0.3ml.After immune, adopting for each experimental animal is observed Raise, drinking-water, whether spirit is normal, whether have poisoning symptom, whether produce allergic reaction, be dead or other abnormal feelings occur Condition.Before immune and after off-test, all animals are weighed.Weighing results carry out statistical analysis.

3 results

3.1 clinical observation

After 3 immune group animal immunes do not observe any allergic reaction or poisoning symptom, the spirit of SPF chickens, adopt feeding, Drinking-water, activity, excrement etc. are all gone well, and do not occur obvious local inflammation equivalent damage clinical side reaction, and occur without death.

3.2 changes of weight

1 is the results are shown in Table, compared with the control, three immune group the weight of animals increase and not notable (the P ＞ of control group difference 0.05), show that immune restructuring bursa of farbricius protein engineering vaccine has no adverse effects to the body weight of animal.

Table 1 recombinates influence of the bursa of farbricius protein engineering vaccine to SPF chicken changes of weight

Lot number	Body weight (g) before immune	10 days body weight (g) after immune	Daily gain (g)	P
					131024	252.17±24.56	426.35±44.78	17.42±2.18	＞ 0.05
131027	244.62±26.33	419.83±42.41	17.57±1.89	＞ 0.05
					131031	255.34±24.92	434.27±39.36	17.89±2.04	＞ 0.05
Control group	260.28±25.75	435.52±44.75	17.53±2.11	-

The animal experiment of embodiment five is grouped and is immunized

1 vaccine is with attacking poison virus

Recombinate bursa of farbricius protein engineering vaccine by Hongqiao Ming Qin research and development centres provide, lot number 131024,131027, The medium virulence live vaccine of 131031, K85 strains is given by Guangdong Yongshun pharmaceutical development center, lot number 2013005.

2 experimental animals

One age in days negative antibody SPF chickens 50, are divided into 5 groups, every group 10.Resisted before immune with ELISA method detection source of parents Body, all test chickens are IBDV negative antibodies, pre- to raise two weeks, adapt to environment.

Two week old SPF immune groups distinguish intramuscular injection protein engineering vaccine and medium virulence live vaccine (K85 strains), 2ml/ Only, 14 days same dose booster immunizations after head exempts from.Take a blood sample once after immune, terminated to 84 days every two weeks, gather 6 times altogether, separation Serum simultaneously preserves in -20 DEG C.

The IgY titres of embodiment six detect

IgY's is horizontal indirect with tradition in the malicious vaccine of IBDV work and vaccine antigen protein immune response, especially serum ELISA method detects.Microwell plate (Nunc Maxisorp, Nalge Nunc International, Denmark) uses antigen egg White or IBDV vaccines (1 μ g/ holes) coating, 2-8 DEG C overnight；5% skim milk closes 1h in 37 DEG C, and antiserum is made into 1: 500,1: 1000,1；2000,1: 4000,1: 8000,1: 16000,1: 32000 times of dilution, at the same it is right as feminine gender by the use of immune PBS serum According to, add microwell plate, 100ul/ holes, 1h be incubated in 37 DEG C, then with rabbit-anti chicken IgY-HRP ELIAS secondary antibody (1: 10000, Sigma, St louis), microwell plate is added, 100ul/ holes, 1h is incubated in 37 DEG C.The colour developing of TAB substrates lucifuge 10min, 2MH₂SO₄ Terminating reaction, absorbance is detected under 450nm wavelength.Using the inverse of highest serum dilution factor as antibody titer, it is average Light absorption value (>=0.2) is higher than the average absorbance value+2SD of preimmune serum, as cutoff values.

As a result

Identical Fluctuation tendency is presented in the IgY antibody titers of four immune group animals, gradually rises, reaches after immune within 42 days Peak, to off-test, antibody level is down to immune latter 14 days level.Three restructuring bursa of farbricius vaccine antigen proteins produce Obvious humoral immune reaction, 42 days antibody is up to 1: 32000, minimum 1: 8000 (see Fig. 7) after being immunized, and IBDV lives malicious epidemic disease Seedling (K85 strains), highest antibody titer are 1: 16000, minimum 1: 8000.It can be seen that the protein-specific of restructuring bursa of fabricius vaccine IgY levels are apparently higher than traditional commercialization living virus vaccine.

The reaction of embodiment septuple group bursa of fabricius vaccine antigen protein and wild poison

Analyzed with reaction of the Westernblot methods to recombinant protein and wild virus infection serum more anti-(1: 2000). Equally, reacted with the street strain and the serum (1: 1000) of restructuring bursa of farbricius vaccine immunity of separation.Rabbit-anti chicken IgY-HRP (1 : 10000) it is used as secondary antibody, the colour developing of DAB substrates.

As a result

Restructuring bursa of fabricius vaccine antiserum has association reaction with VP2 albumen at 56KD, shows that antibody can identify naturally With recombinant protein in 56KD obvious association reaction (see Fig. 6) can also occur for the epitope of wild malicious antigen, wild virus infection antiserum.

The reaction of embodiment eightfold group VP2/3-IL15 albumen and commercialized vaccine

With ELISA method detection recombinant VP 2/3-IL15 albumen and the sero-fast reaction of commercialized vaccine.Concentration is 1ug/ Ml VP2/3-IL15 albumen is coated with 96 hole elisa Plates, and 100 μ l/ holes, 2~8 DEG C are incubated overnight.PBST is washed three times, optimal dilution The K85 strains immune antiboidy of multiple adds ELISA Plate, 100 μ l/ holes, 1h is incubated in 37 DEG C, and PBST is washed three times, rabbit-anti chicken IgY- HRP (1: 10000) secondary antibody adds ELISA Plate, and 100 μ l/ holes are incubated 1h in 37 DEG C, and PBST is washed three times.In addition, K85 vaccine strains Same operation is carried out with recombinant VP 2/3-IL15 protein immunizations antiserum (1: 1000).TAB substrates lucifuge colour developing 10min, 2MH₂SO₄Terminating reaction, absorbance is detected under 450nm wavelength.Using the immune association reaction of VP2/3-IL15 albumen as two The reference of kind analysis.

As a result

The ability ELISA side of the protein induced caused antiserum combination K85 vaccine strains of VP2/3-IL15 of three batches Method is detected.Compared with preimmune control antibody, VP2/3-IL15 protein antiserums show significantly with IBDV K85 epidemic diseases The high response (P ＜ 0.01) (see Fig. 8) of seedling strain.Because IBDV vaccine strains include totivirus, this shows VP2/3-IL15 eggs Antibody caused by white induction can be combined with IBD totivirus.Equally VP2/3-IL15 albumen is detected to IBDV epidemic diseases with ELISA method The sero-fast reaction of seedling.Compared with before being immunized, antiserum caused by the induction of IBDV vaccine strains shows as obvious high response (P ＜ 0.05) (see Fig. 9).It can be seen that IBDV it is vaccine-induced it is more it is anti-can produce cross reaction with VP2/3-IL15 albumen, enter one Step determines the Primary epitope containing VP2 in albumen.

The propagation detection of the splenocyte of embodiment nine

Immune 42 days SPF chickens are put to death and sterile win spleen.Separating Morr. cell and with fresh RPMI1640 culture mediums (Sigma, St.louis) is washed twice.With lysis buffer (0.1M ammonium chlorides) purging twice, red blood cell is removed.Single cell Suspension is put into 96 porocyte plates, and 2 × 10⁵Cell/ml, 100 μ l/ holes, contain 80 μ g/ml gentamicins (Ranbaxy Laboratories) 25mM HEPES (Amersham Pharmacia), 2mM glutamine and 10% hyclone Cultivated in RPMI1640 culture mediums.By the antigen of cell various concentrations：0.1st, 1,5,10,50ug carries out stimulated in vitro per hole, Simultaneously by the use of the ConA in 1ug/ holes as positive control, culture datum hole is as negative control.Cell version is put into 5% CO2 cultures Culture 72h in case (MCO-175SANYO).Cell proliferative conditions are detected with mtt assay (Promega).Breeder reaction stimulus index S.I. represent.All cultures do three repetitions, are as a result expressed as average value.

As a result

In order to evaluate SPF chickens to identification of the recombinant protein vaccine to t cell epitope, Splenocyte Proliferation Assay has been carried out.Point Not Yong VP2/3-IL15 proteantigens and IBDV vaccine antigens to the spleen of VP2/3-IL15 protein engineering seedling and IBDV vaccines is immunized Cell carries out stimulated in vitro, compared with control group, under 10ug/ml concentration, and VP2/3-IL15 albumen and IBDV live seedlings Immune group, generate obvious high-caliber splenic lymphocytic proliferation (S.I.=7.33~9.52) (see Figure 10).Work as VP2/ When the splenocyte of 3-IL15 protein sensitizations is stimulated with vaccine antigen, under 50ug/ml concentration, compared with the control, show as significantly The splenic lymphocytic proliferation (S.I.=6.48~8.03) of (P ＜ 0.01).This shows that the presence of VP2T cell epitopes can produce Strong cell immune response.Positive control ConA groups generate splenocyte in control group and VP2/3-IL15 protein immunization groups Breeder reaction (see Figure 11).

The protest test of embodiment ten

60 two week old SPF chickens are randomly divided into 6 groups, every group 10, immune group intramuscular injection is immunized.1~3 group is exempted from respectively The 20ug/ml recombinant protein engineered vaccines of 3 batches of epidemic disease, 4 groups of intramuscular injection 1ml commercialized vaccine IBDV K85 vaccine strains.5 For group to attack malicious control group (CC), 6 groups non-are attacked malicious control group (NC) to be nonimmune.During 42 age in days, except NC groups, whole immunity tests move Thing is with 10^5.8BLD₅₀/ 0.25ml strong IBDV strains BC6/85 the oral challenges of standard, clinical observation 10 days.If chicken occurs such as Dysentery characterized by white mucous stool, spirit are depressed, and loss of appetite, feather is fluffy, and wing is sagging, close mesh and the clinical symptoms such as doze off, and isolation immediately checks.

The level of protection of different groups is with the ratio of the bursa of farbricius/body weight after slaughtering come (BF/BW), the death rate and the bursa of farbricius Inflammatory reaction represents.Interim BF/BW is the body weight of bursa of farbricius weight × 1000/, every group be expressed as mean+SD (± SD).The death rate of 10 days after poison is attacked to represent with The dead quantity/sum.Histotomy detection is carried out after off-test, as most Whole protection result.Bursal disease variate is evaluated according to bursa of farbricius pathology outward appearance：0 is without lesion, and 1 is Minimal change, and 2 be point Dissipate or the part bursa of farbricius damages, 3 be 50% hair follicles damage, and 4 be 51-75% hair follicles damages, and 5 be that the 76-100% bursa of farbricius damages. Pathology inflammatory reaction value shows unprotected more than 1.Score value ratio is calculated with bursa of farbricius pathology lesion quantity/group total amount.Protection Rate is represented so that bursa of farbricius pathology pathological changes value occurs as 0 and 1 size of animal/group total quantity.

As a result

Immune group animal attacks poison with standard IBDV strains.Immune VP2/3-IL15 protein groups (1~3 group) BF/BW ratios and It is nonimmune it is non-attack malicious group (NC groups, 6 groups) without significant difference (P ＞ 0.05), but apparently higher than attacking malicious control group (5 groups) (P ＜ 0.05), this shows that VP2/3-IL15 albumen provides the protection for attacking poison.Commercialization IBDV K85 groups (4 groups) BF/BW shows and is higher than Malicious control group is attacked, but difference is not notable (P ＞ 0.05) (being shown in Table 2).

All experimental group resistance IBDV attack poison protection and weigh by bursa of farbricius pathology impairment value and (be shown in Table 2).Show IBDV Attack the bursa of farbricius impairment value of 10 days after poison.Recombinant protein engineered vaccine immune group and all chicken show values of NC groups are 0, the bursa of farbricius Without inflammatory reaction, the chicken that IBDV live vaccine groups only have 40% is protected, and lesion numerical value is 1.7, nonimmune to attack malicious group (5 groups) Display infection, average bursa of farbricius pathology pathological changes value is 4.5.

Recombinant protein engineered vaccine immune group chicken whole adaptive immune attacks malicious protection, the death rate 0.But 4 groups are exempted from The epidemic disease commercialization IBDV K85 death rates are 20%, and in terms of bursa of farbricius degree of injury, protection is significantly lower than recombinant protein engineering epidemic disease Seedling immune group (P ＜ 0.01), because living virus vaccine may also cause inflammatory reaction in itself.

Animal challenge test Vaccine effectiveness result is immunized in table 2

Claims (6)

1. a kind of protein engineering vaccine for preventing bursa of Fabricius in poultry, it is characterised in that the major antigen composition of vaccine is engineer Restructuring bursa of farbricius major structural protein B cell epitope, neutralizing epitope, t cell epitope and cell factor fowl interleukin I L-15 The fusion protein of cascaded structure, it is characterised in that specific amino acid sequence is SEQ ID No.2.

A kind of 2. nucleic acid molecules, it is characterised in that fusion protein described in coding claim 1.

3. a kind of carrier, it is characterised in that containing the nucleic acid molecules for having the right to want described in 2.

4. a kind of host cell, it is characterised in that contain the carrier described in claim 3.

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

ctg ggg atg gca cag cca aca caa aac tct gcc gga gca cgg aga agg ccg gag agt cag aaa aca cat gtg aaa agt att tgt ctc cag tac caa ctg tat cta ctt ttg aac agc tat ttc ttt tgc ctt tta aag aat aag act gga cta acc atc ttc ttc cta tgt gct tat gta cca aag aca gaa gca aat cac tgt aag tgg tca gac gtt ctg aaa gat ttg gag ctg atc aag aca tct gaa gac att gac gtc agt tta tat act gca aac aca tac gag gat ata gaa tgc cag gaa cct gta atg aga tgt ttt ttt tta gag atg aaa gtg att ctt cac gaa tgt gat atc aaa aaa tgt agt agg aag cat gat gta cgg aac ata tgg aaa aat gga aat gca aga ttt gca act tac cag ttg aat tcc aca aca gca aaa aaa tgc aaa gaa tgt gaa gag tat gaa gaa aaa aat ttt aca gaa ttt ata cag agt ttt gta aag gtt ata cag agg gaa tgc aga aaa tac gct aac ggt gat atc cca ggt tgc aag aca aac ctg caa gat caa acc caa ggt ggtaca acc gga ccg gcg tcc att ccg gac gac acc cta gag aag cac act ctc agg tca gag acc tcg acc ggt tct ggt ctg cag agc aat ggg aac tac aag ttc ggt ggt gta gca aca tgt gac agc agt gac agg ccc aga atc tac acc ata gct gca gcc aat gat tac caa ttc tca tca cag tac caa gca gat gga ggt tct ggt cca aat cct gaa cta gca aag aac ctg atc aca gaa tac ggc cga ttt gac cca gga gcc atg aac tac aca aaa ttg ata ctg agt gag agg gac cgtctt ggt atc aag acc gta tgg cca aca agg gag tac act gac ttt cgc gag tac ttc atg gag ggt ggt cca tct tgt gac ccc aac gcc cat cgg atg cgt aat ttt ctc gca aac gca cca cag gca ggc agc aag tcg caa aga gcc aag tac ggg aca gca ggc tac gga gtg gag gcc cga ggc ccc aca cca gag gag gca cag agg gaa aaa gac aca cgg atc tca aag aag atg gag ggt tct ggt aat ggg cac agg ggg cct agc ccc ggc cag cta aag tac tgg cag aac aca cga gaa ata cct gat cca aac gag gac tac cta gac tac gtg cat gca gag aag agc cgg ttg gca tca gaa gaa caa atc ggt tct ggt aac cat ggg cgt ggc ccc aac caa gaa cag atg aaa gat ctg ctc ttg act gcg atg gag atg aag cat cgc aat ccc agg cgg gct cca cca aag ccc aag cca aaa ccc aat gct cca aca cag aga ccc cct ggt cgg ctg ggc cgc。

6. fusion protein and pharmaceutically acceptable carrier described in claim 1 are in the protein engineering vaccine of bursa of Fabricius in poultry Application in preparation.