CN104628871A

CN104628871A - Preparation of infectious bursal disease (IBD) protein engineering vaccine

Info

Publication number: CN104628871A
Application number: CN201510072005.XA
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: GUANGZHOU PUTAI BIOTECHNOLOGY Co Ltd
Current assignee: QINGDAO MINGQIN BIOLOGICAL TECHNOLOGY CO., LTD.
Priority date: 2015-02-09
Filing date: 2015-02-09
Publication date: 2015-05-20
Anticipated expiration: 2035-02-09
Also published as: CN104628871B

Abstract

The invention relates to preparation and application of an infectious bursal disease (IBD) protein engineering vaccine. The preparation method comprises the following steps: connecting B cell neutralizing epitope and T cell immunizing epitope of IBDV (infectious bursal disease virus) primary structure proteins VP2 and VP3 as a vaccine frame structure through a flexible linker, connecting with cell factor poultry interleukin 15(chIL-15) in series, cloning into a pRSETB vector, transforming Escherichia coli, fermenting, purifying, emulsifying and the like to obtain the IBD protein engineering vaccine with ideal immunogenicity. The invention also relates to an application method of the vaccine. The animal experiment indicates that the IBD protein engineering vaccine has equivalent effects to the live virus vaccine on the humoral immunity and cellular immunity level, and can stimulate the generation of T lymphopoiesis immunoreaction on the cellular level and the generation of antibody immunoreaction with virus neutralizing activity on the humoral level. The attacking protection test indicates that the IBD protein engineering vaccine group has obviously better effects than the live vaccine group.

Description

A kind of preparation of fabricius bursa protein engineering vaccine of recombinating

Technical field

The invention belongs to biotechnology genetically engineered field, relate generally to the preparation and application of a kind of restructuring fabricius bursa protein engineering vaccine.Particularly, utilize gene recombination technology, the epi-position of major structural protein VP2, VP3 is connected with cytokine fowl IL-15, and be cloned into carrier, transform Host Strains, by fermentation, prepared by purifying, emulsifying process, obtains recombinate fabricius bursa protein engineering vaccine and the application of this vaccine in the avian infectious disease fabricius bursa viral disease of prevention.

Background technology

Infectious bursal disease (Infeetious Bursal Disease, IBD) be that one caused by infectious bursa of Fabricius virus IBDV is acute, high degree in contact sexually transmitted disease, endanger one of poultry husbandry transmissible disease the most serious (Ikuta et al., 2001 at present; Van den Berg, 2000).This virus without cyst membrane, in icosahedron cubic symmetry.Have individual layer capsid, the capsomere that nucleocapsid is 12nm by 32 diameters forms, and diameter is about 60nm.Except complete virus particle, also common have hollow capsid, and in cells infected, virus is normal in lattice-like arrangement.

IBDV genome is made up of two AMPLIGEN sections, be respectively A sections (large sections) and B sections (little sections) (Lombardo et al., 2000), two sections include 5 ' end non-coding region (5 ' non-coding region, 5 '-NCR), coding region and 3 ' end non-coding region (3 '-NCR).IBDV genome A, B sections is encoded 5 kinds of albumen altogether.B segment encodes VP1 albumen (95kDa), connects albumen in virus particle as genome and is called VPg (Delmas et al., 2011, Muller et al., 2003).A sections comprises two partly overlapping ORF, little ORF coding VP5 albumen (17kDa), a large ORF molecular weight of encoding is the polyprotein of 108kDa, this albumen produces VP2 precursor protein pVP2 (48kDa), VP3 (32kDa) by self cleavage, VP4 (28kDa), pVP2 are treated as ripe VP2 (40kDa) further by carboxyl terminal.VP2 and VP3 is structural protein, forms the ectonexine of virion capsid, all containing neutralizing epitope (Azad et al., 1991; Heine and Boyle, 1993; Villegas et al., 2008; Yip et al., 2007).

VP2, by the A sections VP2 genes encoding of IBDV, is the major structural protein of IBDV, accounts for 51% of viral total protein, forms the outer capsid of virus.VP2 and another major structural protein VP3-works the skeleton forming IBDV nucleocapsid.VP2 depends on the protectiveness neutralizing antigenic site of protein tridimensional three-dimensional conformation with IBDV; can induce and produce protectiveness neutralizing antibody; and the neutralizing antibody with its induction of serological specificity protects host passively not by the infection (Lukert and Saif, 2003) of IBDV.VP2 contains conformation dependent (discontinuous) antigenic determinant inducing generation neutralizing antibody of serotype specificity; the neutralizing antibody of its induction can protect host not by the infection of IBDV; therefore VP2 is host protective antigen (Azadetal., 1991 of IBDV.Therefore; it is generally acknowledged that VP2 is the host protective antigen of IBDV; between different strain, the difference of VP2 aminoacid sequence is mainly concentrated and is present in 206th ~ 350 totally 145 amino acids residues; be called VP2 hypervariable region; this region is positioned at (Rautenschlein et al., 2005) between two restriction enzyme site AccI and Spel just.Containing two hydrophilic peak A (210th ~ 225) and peak B (312nd ~ 324) and between region, immediately preceding seven peptide districts of the seven amino acid composition behind the second hydrophilic area, this region of usual title is VP2 hypervariable region, belong to neutralizing epitope, weak poison and strong poison can be identified.First hydrophilic area contributes to the stable of polypeptide conformation, and second identification of hydrophilic area to viral neutralizing monoclonal antibody is relevant.The epitope point that neutralizing monoclonal antibody identifies is a three-dimensional three-dimensional conformation (Coulibaly et al., 2005).Can cause the amino acid variation of epitope point three-dimensional conformation generation subtle change that the antigenicity of IBDV all can be made to change (Letzel et al., 2007, Martinez-Torrecuadrada et al., 2003).The variation of different strain in hypervariable region is comparatively large, the change that the change of molecular structure can cause the change of virus virulence and host to reply vaccine, makes traditional vaccine strain can not control its popular (Pitcovski et al., 2003; Rong et al., 2007).In addition, VP2 causes one of apoptosis principal element (Ikuta et al., 2001 after cell infection IBDV; Van den Berg, 2000).

VP3 albumen is one of main structural protein of IBDV, and molecular mass is about 32kD, accounts for 40% of viral total amount, is positioned at the internal surface of virus particle, plays an important role in the process that IBDV nucleocapsid skeleton is formed.VP3 contains group specific antigen determinant, can induce the generation of neutralizing antibody, but response capacity is extremely weak, and immune protective is not strong.VP3 is necessary for the formation of virus-like particle, and VP3 is required (Lee et al., 2003) for the formation of viral capsid and the complete of form.At least there is the non-overlapped non-neutral epitope of two independence in VP3, one of them is that two serotypes are common, and another is serotype special (Martinez-Torrecuadrada et al., 2003).The VP3 gene of different blood serum subtype IBDV is relatively conservative, and the existence of VP3 is the prerequisite that virus-like particle (VLPs) can correctly be formed, and it is the formation of IBDV capsid and the complete of form is required, and its effect is stable virus RNA.In addition, because VP3 many sections have wetting ability, it is made to play promoter action (Lee et al., 2003) in virus with during antibody response.

Interleukin 15 (interleukin 15, IL.15) be a kind of cytokine recently found, it is similar that much character and the function of IL-15 are considered to same IL-2, as (Grabstein K H, 1994) such as inducing T cell proliferation, the cytotoxicity improving natural killer cell (NK), the B cell proliferation promoting activation and differentiation.The people such as Lillehoj have been cloned into a new gene in cell cdna library, the outer ATG initiator codon of two frames is contained in its 5 ' end non-coding region, proteins encoded contains longer signal peptide (being made up of 66 amino acid), and maturation protein contains the cysteine residues of four high conservatives.This albumen can maintain the lymphocytic growth of chicken T of ConA activation in vitro and strengthen the killing activity of NK cell, this gene and protein feature similarity with the Mammals IL-15 to have reported, determine this genes encoding ChIL-15 (Lillehoj H S etal, 2001) thus.IL-15 is a kind of multi-functional cytokine, comprise the growth that (1) maintains T cell strain CTL-2, inducing cytotoxic T cell is active, the activation of induction pre-sensitization T cell, differentiation, promote GML particularly TCR cytodifferentiation growth, and work in coordination with rapid propagation (Chu C L etal, 1999 that can cause T cell at low concentration with IL-2; Schluns K S etal, 2002) (2) are to normal B cells, after with PMA and anti-IgM pre-activate, IL-15 can induce its proliferative response, when by IL-15 and CD40L combined utilization, B cell IgG secretion (Armitage R T, 1995) can be made; (3) can the differentiation and maturation of induced NK cell, strengthen its killing activity, also significantly can promote NK cell expressing 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 density can increase LPS activation mononuclear macrophage produce proinflammatory inflammation factor as IL-1, IL-6 and TNF α, the IL-15 of lower concentration optionally can suppress the generation of proinflammatory inflammation factor, antagonism inflammatory factor without impact.(6) lymphocyte generation apoptosis is suppressed, and the necrocytosis that IL-2 can be suppressed to mediate (Waldmann T A, 2001).

Traditional deactivation and living virus vaccine inoculate all Zeng Zuowei prevention and control IBDV Main Means.But deactivation vaccine lacks the ability that induction body produces solid immunizing power, especially in cell-mediated immune efficacy.In the face of vvlBDV threatens, deactivation vaccine can not make chicken be protected within whole vegetative period.Due to attenuated IBDVs vaccine often incomplete attenuation, certain pathology damage may be caused to the lymph follicle of the fabricius bursa after immunity, what even have can also cause immunosuppression (Rautenschlein et al., 2005), also the appearance (Snyder, 1990) of high virulence or variant can be caused.And some IBDV variant living vaccine causes immunosuppression and subclinical secondary infection (Fussell, 1998; Donnelly et al., 2000).Therefore, the novel I BDV vaccine developing a kind of safe and effective applicable mass immunization is imperative.

Summary of the invention

The present invention is using major structural protein VP3B cell epitope and VP2B cell Neutralization and crystallization and t cell epitope as vaccine skeleton construction, with avian cytokines interleukin 15 (chIL-15) gene tandem, after expression in escherichia coli, through the technique such as protein purification, emulsification, obtain the recombiant vaccine with Desirable immunogenic.Effective humoral immunization and cell immune response can be induced after this vaccine immunity target animals.

An object of the present invention there are provided a kind of recombinant protein engineered vaccine polypeptide and vaccine composition thereof that can be used for preventing bursa of Fabricius in poultry newly; Two of object of the present invention there are provided structure and the preparation method of described fabricius bursa protein engineering vaccine; Three of object of the present invention there are provided the engineering strain can expressing described fabricius bursa protein engineering vaccine; Four of object of the present invention there are provided the preparation method of described protein engineering vaccine; Five of object of the present invention there are provided the purposes of described fabricius bursa protein engineering vaccine in prevention fabricius bursa.

In first aspect, the invention provides a kind of for preventing restructuring fabricius bursa protein engineering vaccine polypeptide and the composition thereof of fabricius bursa.It contains major structural protein VP3 protein B cell epitope, VP2 protein B cell Neutralization and crystallization and t cell epitope and avian cytokines interleukin 15 (chIL-15).Described fabricius bursa protein engineering vaccine protein or polypeptide or pharmacy acceptable salt and the carrier required for antigen expressed neoepitope Western.Carrier also can comprise the sequence of each epitope of coding separately, and series connection can be undertaken by genetic engineering method.Described vaccine also comprises nonimmune active substance, be the connection portion of each polypeptide, not there is the immunogenicity of epitope, not there is any adjuvanticity yet, mainly contain purification tag, joint peptide, chemically modified part, N end signal peptide and C and hold polyadenylic acid etc.Described pharmacy acceptable salt refers to nontoxicity, stimulation and transformation reactions, is applicable to the salt of human or animal tissues.Inactive substance and pharmacy acceptable salt are well known to those skilled in the art.Restructuring fabricius bursa protein engineering vaccine polypeptid acid sequence is as follows:

LGMAQPTQNSAGARRRPESQKTHVKSICLQYQLYLLLNSYFFCLLKNKTGLTIFFLCAYVPKTEANHCKWSDVLKDLELIKTSEDIDVSLYTANTYEDIECQEPVMRCFFLEMKVILHECDIKKCSRKHDVRNIWKNGNARFATYQLNSTTAKKCKECEEYEEKNFTEFIQSFVKVIQRECRKYANGDIPGCKTNLQDQTQGGTTGPASIPDDTLEKHTLRSETSTGSGLQSNGNYKFGGVATCDSSDRPRIYTIAAANDYQFSSQYQADGGSGPNPELAKNLITEYGRFDPGAMNYTKLILSERDRLGIKTVWPTREYTDFREYFMEGGPSCDPNAHRMRNFLANAPQAGSKSQRAKYGTAGYGVEARGPTPEEAQREKDTRISKKMEGSGNGHRGPSPGQLKYWQNTREIPDPNEDYLDYVHAEKSRLASEEQIGSGNHGRGPNQEQMKDLLLTAMEMKHRNPRRAPPKPKPKPNAPTQRPPGRLGR

In second aspect, the invention provides a kind of nucleic acid molecule, the fabricius bursa protein engineering vaccine polypeptide of its coding described in first aspect present invention.Nucleotide of the present invention can be rna form, DNA form, many epitopes tandem sequence and cytokine sequence is synthesized by synthetic mode, then connect rear clone through genetically engineered operation and enter carrier, be transformed into intestinal bacteria, after screening, fermentation, purifying, obtain fabricius bursa protein engineering vaccine polypeptide.Conventional molecular biology manipulations can be carried out in the present invention to this nucleic acid, as: PCR, digestion with restriction enzyme, connection etc., nucleic acid design 5 ' end and 3 ' end all add restriction enzyme site.Nucleotide sequence in preferred 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 TACGAG 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, it is except containing the coding fabricius bursa protein engineering vaccine nucleotide acid molecule described in second aspect present invention, also containing being connected with this nucleotide sequence is exercisable, at the expression controlling elements needed for procaryotic cell expression (transcribe and translate).The most basic expression controlling elements comprises promotor, transcription terminator, enhanser, selected marker etc., and these controlling elements are known in the art.In the present invention, preferred e. coli bl21 (DE3, Plys) is as expression vector.

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

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

In the 6th, the invention provides a kind of for preventing the restructuring fabricius bursa protein engineering vaccine of bursa of Fabricius in poultry, it comprises polypeptide described in first aspect present invention and pharmaceutically acceptable carrier.Described fabricius bursa protein engineering vaccine can prevent the outburst of bursa of Fabricius in poultry.Pharmaceutically acceptable carrier of the present invention is immunostimulant or immunological adjuvant, and preferred immunological adjuvant is import white-oil adjuvant.

In the 7th, the invention provides the application of the restructuring fabricius bursa protein engineering vaccine described in the 6th aspect.Vaccine can necessarily effective dose intramuscular injection, and intracutaneous or inoculated with subcutaneous injections animal, can produce antibody and the cytokine of q.s, provide antiviral activity, watch for animals and avoid the attack of bursal disease virus epidemic isolates.In addition; in embodiments of the invention; by carrying out vaccine, humoral immunization and cellular immunization detect, target animals attacks malicious simultaneous test, laboratory safety test etc.; show that restructuring fabricius bursa protein engineering vaccine of the present invention is safe (see embodiment four), can watch for animals from bursal disease virus infection (see embodiment five ~ ten).

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

Accompanying drawing explanation

Following accompanying drawing for illustration of specific embodiment of the invention scheme, and is not used in the scope of the invention limiting and defined by claims.Fig. 1 recombinates fabricius bursa protein engineering vaccine expression vector pRSETB-IBDV (VP2/3)-chIL15 design of graphics; Fig. 2 pRSETA-PRRSV (GP5/6/7/Ub) vector plasmid cleavage map, wherein swimming lane 1 is DNAmarker, molecular weight is followed successively by 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp from top to bottom, swimming lane 2 is empty plasmid, swimming lane 3 is plasmid enzyme restriction figure, and swimming lane 4 cuts contrast for non-enzyme; Fig. 3 SDS-PAGE detects figure, wherein 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, and swimming lane 2 is non-induced samples, swimming lane 3 is induced samples, and arrow indication is the target protein of expressing; Fig. 4 Westernblot detects figure, and wherein swimming lane 1 is pre-dyed marker, is followed successively by 97KD, 66KD, 43KD, 31KD, 20KD, 14KD from top to bottom, albumen for the purpose of swimming lane 2.Fig. 5 is that fermented sample SDSPAGE schemes: wherein swimming lane 1 is albumen Marker, be 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 arrow indication is the target protein of expressing; Fig. 6 is restructuring vaccine protein and the how anti-reaction result figure of wild virus infection serum, wherein swimming lane 1 is pre-dyed albumen marker, be followed successively by 116KD, 66KD, 45KD, 35KD, 25KD, 20KD, 10KD from top to bottom, swimming lane 2 is wild poison and recombiant vaccine antiserum(antisera) westernblot result, and swimming lane 3 is restructuring VP2/3-IL15 albumen and open country poison antiserum(antisera) westernblot result; Fig. 7 is that target animals IgY antibody titers detects; Fig. 8 is the association reaction result of immune antiserum(antisera) and IBDV vaccine strain; Fig. 9 is the sero-fast association reaction result of recombinant protein VP2/3-IL15 and living vaccine K85 strain; Figure 10 recombinant VP 2/3-IL15 proteantigen stimulates T cell proliferative response; The proliferative response of Figure 11 K85 vaccine virus antigenic stimulation T cell.

Embodiment

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

The mentality of designing of embodiment one fabricius bursa protein engineering vaccine protein

The present invention is according to current domestic fabricius bursa Major Epidemic strain Viral structural protein VP2, VP3 aminoacid sequence, utilize the strain of relevant biological information software DNASTAR, BIMAS and SYFPEITHI pop to carry out B cell neutralizing epitope and t cell epitope analysis, introduce avian cytokines interleukin 15 (IL-15) as adjuvant molecules simultaneously.By after designed bursal disease virus B cell and t cell epitope and the series connection of interleukin 15 molecular polypeptide in intestinal bacteria coexpression, by fermentation, purifying, the technique such as emulsification, obtain the fabricius bursa protein engineering vaccine with Desirable immunogenic.The vaccine utilizing the present invention to prepare can effectively pre-false proof fabricius bursa.

Comprehensive analysis Guo Neifashi capsule viral prevalence pnca gene group sequence, antigenic structure, Advance of Epidemiological Research, be optimized design to restructuring fabricius bursa protein engineering vaccine.The wetting ability that the present invention utilizes bioinformatics software to carry out its structural protein, antigenicity, plasticity-, the secondary structure of surface accessibility and Garnier-Robson is analyzed, predict on the basis of possible B cell antigen epi-position and t cell epitope, according to the similarity of epitope position and aminoacid sequence, analyze each epidemic isolates and have epitope, and with reference to the sequence information in GenBank, the epitope of prediction is compared, the conservative property of further analysis epitope in different virus strain, thus determine and VP2 structural protein T cell dominant antigen epitope polypeptide 1 section, B cell epi-position 2 sections, VP3 structural protein B cell epi-position 3 sections.By forming the skeleton structure of vaccine after all epi-position series connection, add molecule adjuvant at skeleton nitrogen end simultaneously.The overall structure of this vaccine is:

Molecular Adjuvant(chlL 15)-VP2T Cell Epitope-VP2 B Cell Epitope 1-VP2 B Cell Epitope 2-VP3B Cell Epitope 1-VP3B Cell Epitope 2-VP3B Cell Epitope 3

The structure of embodiment two coli expression carrier and expression strain

The polypeptide-coding nucleotide designed is served the synthesis of extra large handsome biotech company, nucleotide fragments two ends devise BamH I (5 ' end) and HindIII (3 ' end) restriction enzyme site respectively, being cloned on pMD18T carrier after this segment condense respectively, sequencing confirms to insert gene fragment consistent with implementation sequence (see sequence table).By recombinant plasmid called after pMD18T-IBDV (VP2/3)-chIL15 respectively.With corresponding restriction enzyme, two kinds of plasmids are carried out enzyme and cut process, coli expression carrier selects the pRSETB plasmid of Invitrogen company, also identical restriction enzyme ferment treatment is used, enzyme tangent condition: 10 μ l reaction systems, add 2 μ l plasmids in system, restriction enzyme is 5 activity units (New England Biolabs), adds 10 × damping fluid 1 μ l, deionized water polishing, 37 DEG C of enzymes cut 1.5 hours.Enzyme adds 1 μ l 200mM EDTA termination reaction after cutting end.In 1% agarose gel electrophoresis, electrophoresis 30 minutes.Under ultraviolet lamp, 2.86kb pRSETB plasmid and 1467bp IBDV (VP2/3)-IL15 fragment are cut, reclaim test kit specification sheets according to Qiagen company gel and carry out glue recovery.According to carrier: ratio independent and expression vector mixing by multi-epitope nucleotide fragments of fragment 1:2-3, reaction system 15 μ l, connected by T4DNA ligase enzyme, 16 DEG C of connections are spent the night, obtain recombinant plasmid called after pRSETB-IBDV (VP2/3)-chIL15 respectively, (see Fig. 1), transform competent E. coli BL21 (DE3) pLysS.

Transform: pRSETB-IBDV (VP2/3)-chIL15 is put thawed on ice, add 2 μ l Ligature liquid, again mix, ice-water bath 30 minutes, 42 DEG C 30 seconds, then ice bath is put back to rapidly 1.5 minutes, add 1ml LB nutrient solution, 37 DEG C, quiescent culture 1 hour, 4000g low-temperature centrifugation 10 abandons supernatant second, with the resuspended thalline of 200 μ lLB substratum; Bacterium liquid is spread evenly across on the LB agar plate containing 100 μ g/mL penbritins, is inverted in 37 DEG C of thermostat containers and cultivates 12-16 hour, until Clone formation.

Qualification: the mono-clonal on picking flat board is in LB substratum, 37 DEG C, 200rpm shakes cultivation 12 hours, extract plasmid, restriction endonuclease BamH I and HindIII is used to carry out double digestion respectively, can cut out the clone of corresponding bursa of fabricius vaccine gene size fragment, 1467bp, tentatively can be defined as positive colony (see Fig. 2); Positive colony carries out determined dna sequence and verifies its exactness (see sequence table) further.

Abduction delivering.By positive colony incubated overnight, morning next day, by 1: 100 switching, cultivates after 3 hours, adds 0.2mM IPTG, continues cultivation 4 hours, prepares sample; Conventional SDS-PAGE testing goal protein expression situation---at 56KD (see Fig. 3), see specific band for correct clone; Get correct clone, amplification culture, SDS-PAGE confirms to express correctly, and it expresses accuracy (see Fig. 4) to use conventional Western-blot to confirm further; After above-mentioned structure and qualification program, the positive colony selected can be carried out the foundation of original species word bank as engineering bacteria, bacterial classification name pRSETB-IBDV (VP2/3)-IL15/BL21 (DE3, Plys).

The fermentation of embodiment three engineering bacteria, purifying and emulsification

Production bacterial classification is got in fermentation, is inoculated in (containing 100 μ g/ml penbritins) in 2ml LB liquid nutrient medium, 37 DEG C, 200rpm shaking culture 12 hours activated spawn.Access shaking flask with the inoculum size of 1: 100 again, 37 DEG C of shaking culture, to OD600=3, can be inoculated into fermentor tank in 10% ratio.Fermentation substratum is semisynthetic medium, with distilled water preparation, wherein not containing any microbiotic.Correct dissolved oxygen and pH value electrode, open tank body and stir, revolution is 300rpm, the online sterilizing of tank body, when culture-liquid temp is down to 37.0 DEG C in tank, demarcates pH and dissolved oxygen (OD) zero point.Leavening temperature is 37.0 ± 0.1 DEG C, dissolved oxygen controls about 40%, pH controls 7.0, flow feeding 500ml during cultivation thalline OD600=1.0 ~ 1.2 after inoculation, within after feed supplement 1 hour, add IPTG (final concentration is 0.2mM) abduction delivering, continuous induction secondary fermentation in 5 hours terminates, and sampling is SDS-PAGE and is examined and determine expression (see Fig. 5).

The thalline that purifying will be collected, ultrasonic with carrying out after occlusion body washing lotion I (1%Triton X-100,20mMTris-cl PH8.0) suspendible, 2000W ultrasonic degradation 1 hour.4 DEG C, 12000rpm collected by centrifugation occlusion body, and wash occlusion body with occlusion body washing lotion II (1%DOC, 4M urea, 20mMTris-cl PH8.0) suspendible twice ultrasonic, secondary low-temperature centrifugation collects occlusion body.Occlusion body precipitation uses 8M urea, and 0.3% β-ME, 20mM Tris-cl (pH=8.00) mix, and stirring at room temperature 4 hours, 8000rpm low-temperature centrifugation 30min, discards precipitation.Metaprotein 1: 100 dilutes, and renaturation solution Tris (PH8.0) buffer system, adds 0.3M arginine, and 4 DEG C are stirred renaturation 24 hours.The 20mM phosphoric acid buffer of renaturation solution pH=8.0,0.5M sodium-chlor, 20mM imidazoles, affinity column in balance, with the 20mM phosphoric acid buffer of pH=8.0,0.5M sodium-chlor, 0.5M imidazoles wash-out; Fabricius bursa protein engineering of must recombinating vaccine work in-process stoste.

The PBS of the work in-process sterilizing of purifying is diluted to 100 μ g/ml by emulsification.Follow the example of the Montanide ISA 50V2 adjuvant of Guo Sai BIC Corp, through 121 DEG C, sterilizing 15 minutes, for subsequent use.By oil phase: aqueous phase=50: the proportions of 50, first oil phase is added in emulsion tank, start stirrer and slowly stir with the speed of 80-100r/min, slowly add aqueous phase, stir 2min again after adding, then with 5500r/min high-speed circulating emulsification 9min, make the single-phase vaccine of water-in-oil.

Embodiment four recombinate the fabricius bursa protein engineering vaccine safety test

1 experimental animal 30 age in days SPF chicken 20 plumage.

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

Test adopts the test design of single-factor completely random, at random 20 30 age in days experimental animals is divided into 4 groups at random, 3 lot number groups and control group, often organizes 10, without weight differences between group.Immune group, in every chicken leg portion intramuscular injection vaccine 0.3ml/ head, is observed to 10 days, and control group uses physiological saline emulsion 0.3ml.After immunity, observe that adopting of each laboratory animal is raised, drunk water, spirit whether normally, whether have toxicity symptom, whether produce anaphylaxis, whether dead or occur other abnormal conditions.Before immunity and after off-test, all animals is weighed.Weighing results carries out statistical analysis.

3 results

3.1 clinical observation

All do not observe any anaphylaxis or toxicity symptom after 3 immune group animal immunes, the spirit of SPF chicken, adopt raise, drink water, activity, all are normal for ight soil etc., there is not obvious local inflammation equivalent damage clinical side reaction, and occur without death.

3.2 body weight change

The results are shown in Table 1, compared with the control, three immune group the weight of animals increase and control group difference remarkable (P > 0.05), show that the body weight of immunity restructuring fabricius bursa protein engineering vaccine to animal has no adverse effects.

Table 1 recombinates fabricius bursa protein engineering vaccine to the impact of SPF chicken body weight change

Lot number	Body weight (g) before immunity	Immunity latter 10 days body weight (g)	Day weight 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 grouping of embodiment five animal experiment and immunity

1 vaccine is with to attack poison viral

Restructuring fabricius bursa protein engineering vaccine is provided by Hongqiao Ming Qin research and development centre, and lot number is that 131024,131027,131031, K85 strain mesogenic living vaccines are so kind as to give by pharmaceutical development center, Yongshun, Guangdong, and lot number is 2013005.

2 laboratory animal

One age in days negative antibody SPF chicken 50, is divided into 5 groups, often organizes 10.The front ELISA method of immunity detects maternal antibody, and all test chickens are IBDV negative antibody, raise two weeks in advance, conform.

SPF immune group in two week age intramuscular injection protein engineering vaccine and mesogenic living vaccine (K85 strain) respectively, only, head exempts from latter 14 days same dose booster immunizations to 2ml/.After immunity, blood sampling in every two weeks once, terminated to 84 days, altogether collection 6 times, separation of serum in-20 DEG C of preservations.

Embodiment six IgY titre detects

IBDV malicious vaccine alive and vaccine antigen protein immune response, especially in serum, the level of IgY detects with traditional indirect ELISA method.Microwell plate (Nunc Maxisorp, Nalge Nunc International, Denmark) antigen protein or IBDV vaccine (1 μ g/ hole) wrap quilt, and 2-8 DEG C is spent the night; Antiserum(antisera), in 37 DEG C of closed 1h, is done 1: 500,1: 1000,1 by 5% skimmed milk; 2000,1: 4000,1: 8000,1: 16000,1: 32000 times of dilution, uses the serum of immune PBS as negative control simultaneously, add microwell plate, 100ul/ hole, hatch 1h in 37 DEG C, then by the ELIAS secondary antibody (1: 10000 of the anti-chicken IgY-HRP of rabbit, Sigma, St louis), add microwell plate, 100ul/ hole, hatches 1h in 37 DEG C.The colour developing of TAB substrate lucifuge 10min, 2MH ₂sO ₄termination reaction, detects absorbance under 450nm wavelength.Using the dilution inverse of highest serum as antibody titers, its average absorbance value (>=0.2) higher than the average absorbance value+2SD of preimmune serum, as cutoff value.

Result

The IgY antibody titers of four immune group animals presents identical Fluctuation tendency, raises gradually after immunity, within 42 days, reaches maximum, and to off-test, antibody horizontal is down to the immunity level of latter 14 days.Three restructuring fabricius bursa vaccine antigen proteins produce obvious humoral immune reaction, immunity latter 42 days antibody can reach 1: 32000, and minimum is 1: 8000 (see Fig. 7), and IBDV malicious vaccine (K85 strain) alive, most antibody titers is 1: 16000, and minimum is 1: 8000.The protein-specific IgY level of visible restructuring bursa of fabricius vaccine is apparently higher than traditional commercialization living virus vaccine.

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

Analyze with the reaction of Westernblot method to recombinant protein and wild virus infection serum many anti-(1: 2000).Equally, react with the street strain be separated and the serum (1: 1000) of restructuring fabricius bursa vaccine immunity.The anti-chicken IgY-HRP (1: 10000) of rabbit resists as two, and DAB substrate develops the color.

Result

Restructuring bursa of fabricius vaccine antiserum(antisera) and VP2 albumen have association reaction at 56KD place, and show that antibody can identify the epi-position of the malicious antigen in natural open country, wild virus infection antiserum(antisera) and recombinant protein at 56KD, obvious association reaction (see Fig. 6) can also occur.

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

Recombinant VP 2/3-IL15 albumen and the sero-fast reaction of commercialized vaccine is detected by ELISA method.Concentration be 1ug/ml VP2/3-IL15 albumen bag by 96 hole enzyme plates, 100 μ l/ holes, 2 ~ 8 DEG C of night incubation.PBST washs three times, and the K85 strain immune antibody of optimum diluting multiple adds enzyme plate, 100 μ l/ holes, hatch 1h in 37 DEG C, PBST washs three times, and the anti-chicken IgY-HRP (1: 10000) of rabbit two is anti-adds enzyme plate, 100 μ l/ holes, hatch 1h in 37 DEG C, PBST washs three times.In addition, K85 vaccine strain carries out same operation with recombinant VP 2/3-IL15 protein immunization antiserum(antisera) (1: 1000).The colour developing of TAB substrate lucifuge 10min, 2MH ₂sO ₄termination reaction, detects absorbance under 450nm wavelength.Using the reference that the immune association reaction of VP2/3-IL15 albumen is analyzed as two kinds.

Result

The antiserum(antisera) of the protein induced generation of VP2/3-IL15 of three batches detects in conjunction with the ability ELISA method of K85 vaccine strain.Compared with preimmune control antibody, VP2/3-IL15 protein antiserum shows significantly and the hyperergy (P < 0.01) (see Fig. 8) of IBDV K85 vaccine strain.Because IBDV vaccine strain comprises totivirus, this shows that the antibody of the protein induced generation of VP2/3-IL15 can be combined with IBD totivirus.VP2/3-IL15 albumen is detected to the sero-fast reaction of IBDV vaccine equally by ELISA method.Compared with before immunity, the antiserum(antisera) that the induction of IBDV vaccine strain produces shows as obvious hyperergy (P < 0.05) (see Fig. 9).Vaccine-induced how anti-of visible IBDV can produce cross reaction with VP2/3-IL15 albumen, further determined that the Primary epitope containing VP2 in albumen.

The propagation of embodiment nine splenocyte detects

Immunity 42 days SPF chickens are put to death and asepticly wins spleen.Separating Morr. cell also washes twice with fresh RPMI1640 substratum (Sigma, St.louis).With lysis buffer (0.1M ammonium chloride) purge twice, removing red corpuscle.Single cell suspension puts into 96 porocyte plates, and 2 × 10 ⁵cell/ml, 100 μ l/ holes, cultivate in the RPMI1640 substratum containing 80 μ g/ml gentamicin (Ranbaxy Laboratories) 25mM HEPES (Amersham Pharmacia), 2mM glutamine and 10% foetal calf serum.Antigen by cell different concns: 0.1,1,5,10, the every hole of 50ug carries out stimulated in vitro, use the ConA in 1ug/ hole as positive control simultaneously, cultivate datum hole as negative control.72h cultivated by the CO2 incubator (MCO-175SANYO) cell version being put into 5%.Cell proliferative conditions is detected with mtt assay (Promega).Proliferative response stimulation index S.I. represents.All cultures do three repetitions, and result is expressed as mean value.

Result

In order to evaluate SPF chicken to the identification of recombinant protein vaccine to t cell epitope, carry out Splenocyte Proliferation Assay.With VP2/3-IL15 proteantigen and IBDV vaccine antigen, the splenocyte to immune VP2/3-IL15 protein engineering seedling and IBDV vaccine carries out stimulated in vitro respectively, compared with control group, even if under the concentration of 10ug/ml, VP2/3-IL15 albumen and IBDV seedling immune group alive, all create obvious high-caliber splenic lymphocytic proliferation (S.I.=7.33 ~ 9.52) (see Figure 10).When the splenocyte vaccine antigen of VP2/3-IL15 protein sensitization stimulates, under 50ug/ml concentration, compared with the control, the splenic lymphocytic proliferation (S.I.=6.48 ~ 8.03) of significantly (P < 0.01) is shown as.This shows that the existence of VP2T cell epitope can produce strong cell immune response.Positive control ConA group all creates splenic lymphocytic proliferation (see Figure 11) at control group and VP2/3-IL15 protein immunization group.

Embodiment ten protest test

By 60 two week age SPF chicken be divided into 6 groups at random, often organize 10, immune group intramuscular injection immunity.The 20ug/ml recombinant protein engineered vaccine of 1 ~ 3 group of respectively immunity 3 batches, 4 groups of intramuscular injection 1ml commercialized vaccine IBDV K85 vaccine strains.5 groups for attacking malicious control group (CC), 6 groups is nonimmunely non-ly attack malicious control group (NC).During 42 age in days, except NC group, whole immunity test animal is with 10 ^5.8bLD ₅₀the standard strong IBDV strain BC6/85 oral challenge of/0.25ml, clinical observation 10 days.If chicken occurs that such as dysentery characterized by white mucous stool, spirit are depressed, appetite declines, and feather is fluffy, and wing is sagging, closes order and the clinical symptom such as to doze off, isolate inspection immediately.

The level of protection of different group is come (BF/BW), mortality ratio and fabricius bursa Inflammatory response with the ratio of the fabricius bursa/body weight after slaughtering and is represented.Interim BF/BW is fabricius bursa weight × 1000/ body weight, often group be expressed as mean+SD ( ± SD).Attack the poison mortality ratio of latter 10 days to represent with dead quantity/sum.Tissue slice detects and carries out after off-test, as final protection result.Fabricius bursa variate is evaluated according to fabricius bursa pathology outward appearance: 0 is that 1 is Minimal change without pathology, and 2 is dispersion or the damage of the part fabricius bursa, and 3 is 50% hair follicles damage, and 4 is 51-75% hair follicles damage, and 5 is the damage of the 76-100% fabricius bursa.Pathology Inflammatory response value is greater than 1 and shows not protect.Score value ratio calculates with fabricius bursa pathology pathology quantity/group total amount.Protection ratio with occur fabricius bursa pathology pathology value be 0 and 1 size of animal/group total quantity represent.

Result

Immune group animal standard I BDV strain attacks poison.Immunity VP2/3-IL15 protein groups (1 ~ 3 group) BF/BW ratio and nonimmunely non-ly attack malicious group of (NC group; 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 of attacking poison.Commercialization IBDV K85 group (4 groups) BF/BW shows higher than attacking malicious control group, but difference is not significantly (P > 0.05) (see table 2).

All experimental group opposing IBDV are attacked poison protection and are weighed (see table 2) by fabricius bursa pathology impairment value.Show that IBDV attacks the poison fabricius bursa impairment value of latter 10 days.Recombinant protein engineered vaccine immune group and all chicken displayed values of NC group are 0; the fabricius bursa is without Inflammatory response, and IBDV living vaccine group only has the chicken of 40% to obtain protection, and pathology numerical value is 1.7; nonimmune attack malicious group (5 groups) display infect, average fabricius bursa pathology pathology value is 4.5.

The whole adaptive immune of recombinant protein engineered vaccine immune group chicken attacks poison protection, and mortality ratio is 0.But; 4 groups of immunity commercialization IBDV K85 mortality ratio are 20%; from fabricius bursa degree of injury, protection is starkly lower than recombinant protein engineered vaccine immune group (P < 0.01), because living virus vaccine itself also may cause Inflammatory response.

Table 2 immune animal challenge test Vaccine effectiveness result

Claims

1. a fabricius bursa protein engineering vaccine fusion rotein, its aminoacid sequence is SEQ ID No.2.

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

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

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

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

ctg ggg atg gca cag cca aca caa aac tct gcc gga gca cgg aga agg ccg gag agt cag aaa aca cat gtgaaa 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 ctaacc atc ttc ttc cta tgt gct tat gta cca aag aca gaa gca aat cac tgt aag tgg tca gac gtt ctg aaa gat ttggag ctg atc aag aca tct gaa gac att gac gtc agt tta tat act gca aac aca tac gag gat ata gaa tgc caggaa cct gta atg aga tgt ttt ttt tta gag atg 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 act tac cag ttg aat tcc aca aca gca aaaaaa tgc aaa gaa tgt gaa gag tat gaa gaa aaa aat ttt aca gaa ttt ata cag agt ttt gta aag gtt ata cag agggaa tgc aga aaa tac gct aac ggt gat atc cca ggt tgc aag aca aac ctg caa gat caa acc caa ggt ggt acaacc gga ccg gcg tcc att ccg gac gac acc cta gag aag cac act ctc agg tca gag acc tcg acc ggt tct ggtctg cag agc aat ggg aac tac aag ttc ggt ggt gta gca aca tgt gac agc agt gac agg ccc aga atc tac accata gct gca gcc aat gat tac caa ttc tca tca cag tac caa gca gat gga ggt tct ggt cca aat cct gaa ctagca aag aac ctg atc aca gaa tac ggc cga 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 act gac ttt cgc gag tacttc atg gag ggtggt cca tct tgt gac ccc aac gcc cat cgg atg cgt aat ttt ctc gca aac gca cca cag gca ggc agc aag tcgcaa aga gcc aag tac ggg aca gca ggc tac gga gtg gag gcc cga ggc ccc aca cca gag gag gca cagagg gaa aaa gac aca cgg atc tca aag aag atg gag ggt tct ggt aat ggg cac agg ggg cct agc ccc ggccag cta aag tac tgg cag aac aca cga gaa ata cct gat cca aac gag gac tac cta gac tac gtg cat gca gagaag agc cgg ttg gca tca gaa gaa caa atc ggt tct ggt aac cat ggg cgt ggc ccc aac caa gaa cag atg aaagat ctg ctc ttg act gcg atg gag atg aag cat cgc aat ccc agg cgg gct cca cca aag ccc aag cca aaa cccaat gct cca aca cag aga ccc cct ggt cgg ctg ggc cgc 。

6., for preventing a vaccine for bursa of Fabricius in poultry, it comprises fusion rotein according to claim 1 and pharmaceutically acceptable carrier.

7. the application of fusion rotein according to claim 1 in preparation restructuring fabricius bursa protein engineering vaccine.