CN100475963C - Chicken infectivity bursa of Fabricius virus VP2 cDNA, its expression vector, expressed recombinant protein and application thereof - Google Patents
Chicken infectivity bursa of Fabricius virus VP2 cDNA, its expression vector, expressed recombinant protein and application thereof Download PDFInfo
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Abstract
The invention discloses a novel chicken infectious bursal disease virusVP 2c DNA (SEQ ID NO: 1), construction of its expression carrier, expressed recombined VP2 protein and the application of said recombined protein in preparing subunit genetic engineering vaccine against chicken infection bursal disease virus. The chicken infectious bursal disease virusVP 2c DNA can be highly expressed in yeast cell, and expressed recombined protein possesses biological activity and immunogenicity of the chicken infectious bursal disease virus natural protein . The expressed recombined protein can be produced into vaccine, and it is demonstrated through immunity chicken test that: the protein subunit vaccine can effectively induce body to generate spcial humoral immune response, make immunity chicken get 90% protection from deadly attack of highly pathogenic vv IBDV, and effectively prevent virus proliferation in the body.
Description
Technical field
The present invention relates to a kind of cDNA, relate in particular to the structure of a kind of engineered chicken infectivity bursa of Fabricius virus VP 2 cDNA, its expression vector, expressed recombinant VP 2 albumen and the application of this recombinant protein in the anti-bursal disease vaccine of preparation.
Background technology
Infectious bursal disease is to cause the immunosuppressant important virus disease of chicken body, and the poultry husbandry in the world in serious harm.Traditional vaccine plays important effect in the history of anti-this disease of system, new variation-serotype the variant that presents in recent years along with this disease, the appearance of highly virulent strain, feasible more and more thorny to the anti-system of this disease, some is difficult to catch up with the variation of this disease to the development speed of traditional vaccine.Development of new can prevent that effectively system makes a variation and the requirement of the new generation vaccine of highly virulent strain is particularly urgent fast.The protein subunit recombinant vaccine is to remove genetic material but the safe vaccine of reservation virus antigenicity, does not have the danger of the poison that looses.This developing vaccines and use and will provide effective instrument and means for the infectious bursal disease of anti-system current popular, and can be anti-system bird immunity and suppress disease effective reference and experience are provided.
Methanol yeast (Pichia pastoris) expression system is an eukaryotic expression system that grew up in recent years, it is not only easy and simple to handle as prokaryotic organism, yeast cell is cultivated easily, it is quick to grow, with low cost, be suitable for the heavy industrialization fermentative production, and can carry out correct folding and translation post-treatment to recombinant protein and modify, as glycosylation, methylate and beta sheet etc.Yeast expression system is that expression amount is the highest in all expression systems, can reach more than the grams per liter, and the expression amount that has is up to 12 grams per liters.Yeast expression carrier has secretor type and nonsecreting type, and secretor type can be with expressed protein excretion in liquid nutrient medium, and the excretory albumen of yeast own is considerably less, and foreign protein is considerably less, is easy to purifying.Pichiapastoris has and can utilize methyl alcohol as special advantages such as the carbon source and the energy, is considered to a kind of outstanding engineering bacteria of secreting, expressing or non-secreting expression of exogenous gene.
Usefulness different carriers such as Jagadish have been expressed the big fragment of IBDV in yeast, the result shows that the processing of polyprotein after translating or in the translation process with non-fusion protein form expression can produce correct VP3, but detect less than VP2.But the amyloid protein precursor with fusion protein form expression can produce stable VP2 and VP3, illustrates that the N-terminal at the polyprotein of expressing needs the protection of part Yeast protein sequence, otherwise may be by protease hydrolysis.The big fragment of IBDV that Macreadie etc. have used yeast expression.With injection SPF chicken after yeast lysate 100-200 μ L (being equivalent to 50 μ g VP2) that transforms and the Freund's incomplete adjuvant emulsification, can stimulate generation very high ELISA antibody and neutralizing antibody.With serum abdominal injection 1 Japanese instar chickling of 1mL immunity chicken, used 100CID in second day
50IBDV attack poison, detect fabricius bursa toxic amount after three days.The result shows that immune chicken serum can provide good protection to chick.Fahey etc. with yeast expression IBDV VP2, make immunity SPF chicken in 10 age in week behind the oily adjuvant seedling.Immunity 2 Zhou Houke detect antibody, continue then to rise, and immunity back 6 all ELISA antibody titerss reach the peak, and NAT peaked after 8 weeks.
Studies show that with the IBDV VP2 immunity of yeast expression, its effect is the same with traditional vaccine.Compared with the conventional I BD vaccine with SPF chicken bursa or cell culture preparation, saccharomycetic cultivation is simple, convenient, and required substratum price is also lower, and expression product can be used through slightly carrying.Therefore, the IBD subunit seedling with yeast expression production will have the better application prospect.
The expression efficiency of original IBDV VP2 gene in yeast is not high, need carry out molecular modification and modification to it, to improve its expression efficiency in yeast cell, further make anti-infectious bursal disease protein subunit vaccine with resulting recombinant VP 2 albumen.
Summary of the invention
The present invention's technical problem at first to be solved is to overcome the deficiencies in the prior art, chicken infectivity bursa of Fabricius virus VP 2 cDNA is carried out molecular modification and modification, provide a kind of chicken infectivity bursa of Fabricius virus that is adapted at efficiently expressing in the yeast cell artificial VP2 cDNA.
The present invention's technical problem at first to be solved realizes by following technological approaches:
The artificial VP2cDNA of a kind of chicken infectivity bursa of Fabricius virus that is adapted at efficiently expressing in the yeast cell, it has the nucleotide sequence shown in the SEQ ID NO:1.
The present invention is under the prerequisite that does not change the coded aminoacid sequence of original chicken infectivity bursa of Fabricius virus VP 2 cDNA, selection deflection according to the pichia spp codon is optimized the chicken infectivity bursa of Fabricius virus VP 2 cDNA codon, evade some and may reduce the sequence of expression, obtained a kind of new artificial VP2 cDNA of the chicken infectivity bursa of Fabricius virus that is adapted at efficiently expressing in yeast cell sequence.
The present invention will solve the clone that second technical problem provides the recombinant yeast expression vector of nucleotide sequence shown in a kind of SEQ of containing ID NO.1 and contain this recombinant yeast expression vector.
The present invention will solve second technical problem and realize by following technological approaches:
A kind of recombinant yeast expression vector contains the nucleotide sequence shown in the SEQ ID NO.1.Recombinant yeast expression vector of the present invention can make up by the ordinary method of this area and form, promptly just the nucleotide sequence shown in the SEQID NO.1 is inserted between the suitable restriction enzyme site of Yeast expression carrier, makes that the nucleotide sequence shown in the SEQ ID NO:1 is exercisable to be connected with the yeast cell to express regulating and controlling sequence.As the most preferred embodiment of the present invention, be preferably the nucleotide sequence shown in the SEQ ID NO:1 is inserted between the XholI and XbaI restriction enzyme site of Yeast expression carrier pPICZaC, make this nucleotide sequence be positioned at the downstream of AOX1 promotor and regulated and control by it, obtain recombinant yeast expression vector.
The constructed recombinant yeast expression vector of the present invention can be by conventional method transformed host cell, and described host cell can be pichia spp cell (Pichic pastoris), cerevisiae (Saccharomyces cerevisiae) or saccharomyces lactis cell (Hansenula polymorpha).As the most preferred embodiment of the present invention, be preferably the method that adopts electricity to transform recombinant yeast expression vector and transform SMD1168 pichia spp strain (SMD1168 Pichia strain).
The present invention will solve the 3rd technical problem and provide a kind of chicken infectivity bursa of Fabricius virus recombinant VP 2 albumen, and this recombinant protein has the biologic activity and the immunogenicity of chicken infectivity bursa of Fabricius virus native protein.
The present invention will solve the 3rd technical problem and realize by following technological approaches:
A kind of chicken infectivity bursa of Fabricius virus recombinant VP 2 albumen, this recombinant VP 2 albumen are mainly coded by the nucleotide sequence shown in the SEQID NO:1.
The 4th technical problem to be solved by this invention provides the proteic method of a kind of preparation chicken infectivity bursa of Fabricius virus recombinant VP 2.
The 4th technical problem to be solved by this invention realizes by following technological approaches:
The proteic method of a kind of preparation chicken infectivity bursa of Fabricius virus recombinant VP 2 may further comprise the steps:
Cultivate usefulness recombinant yeast expression vector of the present invention institute transformed yeast cells, induce the proteic expression of recombinant VP 2, reclaim and the expressed recombinant VP 2 albumen of purifying.
In the proteic method of above-mentioned preparation recombinant VP 2, preferred, described yeast cell is SMD1168 pichia spp strain (SMD1168 Pichia strain).
Another technical problem to be solved by this invention provides a kind of protein subunit recombinant vaccine, but this genetic engineering subunit vaccine effectively preventing infectious bursal disease.
Another technical problem to be solved by this invention realizes by following technological approaches:
A kind of protein subunit recombinant vaccine of anti-infectious bursal disease, this vaccine contain the recombinant VP 2 albumen of the present invention that effective dose is gone up in immunity.
In order to reach better immune effect, protein subunit recombinant vaccine of the present invention also can contain pharmaceutically acceptable carrier or excipient etc.
The integral body of an optimized technical scheme of the present invention is described:
Use the isolating voluntarily domestic chicken infectivity bursa of Fabricius virus highly virulent strain genome A fragment of RT-PCR technology amplification, after the gene sequencing, application software DNAstar has a liking for table partially according to the pichia spp codon IBDV VP2 genes encoding is carried out molecular modification and modification, obtain adapting to the new gene order of in the pichia spp cell, expressing, behind new gene order synthetic, it is cloned into the T carrier, and subclone is gone into Yeast expression carrier then.With electric method for transformation transformed yeast competent cell, antibiotic-screening, PCR identifies, obtains restructuring yeast strains.Restructuring yeast strains is carried out the small-scale abduction delivering, and screening obtains the high expression level bacterial strain of stably express, and it is domesticated for engineering strain.Engineering bacteria is induced on a large scale, and expression amount reaches the level of 0.4g/L.The recombinant protein that detects the proof expression through agar diffusion test (AIDT), SDS denaturing polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot has the biologic activity and the immunogenicity of chicken infectivity bursa of Fabricius virus native protein.
Chicken infectivity bursa of Fabricius virus VP 2 cDNA of the present invention can efficiently express (level that expression amount reaches 0.4g/L) in yeast cell, expressed recombinant protein has the biologic activity and the immunogenicity of chicken infectivity bursa of Fabricius virus native protein, expressed recombinant protein is made subunit vaccine, immunity chicken experimental result shows: this infectious bursal disease subunit vaccine can effectively induce body to produce specific humoral immunoresponse(HI), the guarantor of the opposing highly pathogenicity vvIBDV lethal hit of immune chicken acquisition 90% is expanded, and can effectively stop virus propagation in vivo, the experimental result explanation, protein subunit recombinant vaccine of the present invention can effectively be prevented and treated infectious bursal disease.
With the recombinant VP 2 protein immunization of yeast expression, its effect is the same with traditional vaccine.Compared with the conventional I BD vaccine with SPF chicken bursa or cell culture preparation, saccharomycetic cultivation is simple, convenient, and required substratum price is also lower, and expression product can be used through slightly carrying.Therefore, protein subunit vaccine of the present invention is compared with currently available vaccines, existing vaccines and will be had the better application prospect.
The protein subunit recombinant vaccine is to remove genetic material but the safe vaccine of reservation virus antigenicity, does not have the danger of the poison that looses.Subunit vaccine of the present invention will provide effective instrument and means for the infectious bursal disease of anti-system current popular, and can be anti-system bird immunity and suppress disease effective reference and experience are provided, can effectively prevent the loss that eqpidemic disease causes after dropping into application, also reduced simultaneously aquaculture cost, have important scientific meaning and using value, and can bring better economic benefit and social benefit.
The usage of protein subunit recombinant vaccine of the present invention and consumption:
Usage: via intramuscular injection.
Consumption: chick 0.3mL/ plumage, the chicken 0.5mL/ plumage of growing up
Description of drawings
Fig. 1 chicken infectivity bursa of Fabricius virus VP 2 gene and vector construction collection of illustrative plates.
The segmental RT-PCR result of Fig. 2 IBDVA.
M, dna molecular amount standard DL15000+DL2000 (TaKaRa company); 1, IBDV OF243 gene 3.1Kb; 2, ultrapure water is template PCR contrast.
Fig. 3 restructured Pichia pastoris in expression carrier PCR qualification result.
1,2, IBDV VP2 gene 1.3Kb; M, 1Kb dna molecular amount standard (MBI company).
Fig. 4 yeast recon qualification result.
1-6, be Mut
+The strain of phenotype recombination yeast; M, 1Kb dna molecular amount standard (NEB company).
Fig. 5 recombination yeast strain inducible protein Western blot result.
1, pPICZ α C/SMD1168 empty carrier recombination microzyme contrast; 1-7 is homophyletic pPICZ α VP2/SMD1168 recombination microzyme secretory protein not; M, molecular weight of albumen standard.
Further describe preparation method of the present invention and beneficial effect by the following examples, it should be understood that these embodiment only are used for the purpose of illustration, never limit protection scope of the present invention.
Embodiment
Material and reagent
1 viral chicken infectivity bursa of Fabricius virus virulent Gx strain is separated by inventor laboratory.
2 bacterial classifications and carrier competent escherichia coli cell DH5a are by this making in laboratory.Pichia spp bacterial classification SMD1168 and carrier pPICZaC are available from American I nvitrogen company, and pMD18-T is available from Dalian TaKaRa biotech firm.
3 test reagent ThermoScript II superscript
TMII is available from Invitrogen company, and ExpandHigh Fidelity PCR System is available from German Roche company, and other reagent are analytical pure.Sepharose reclaims test kit available from Shanghai China Shun biotech firm.
Synthetic and the clone of [embodiment 1] reorganization chicken infectivity bursa of Fabricius virus VP 2 cDNA
The extraction of 1 viral RNA is ground the SPF chicken bursa of infected chicken very virulent infectious bursal disease virus Gx (vvIBDV-Gx) strain with mill.Get 200 μ l pathological material of diseases and add TE (PH8.0), add 5 μ l Proteinase Ks and 10%SDS 50 μ l, 56 ℃ of water-baths 3 hours to 500 μ l.Add equal-volume phenol/chloroform extracting 3 times, the extracting of equal-volume chloroform once.Shift out supernatant to another 1.5ml centrifuge tube, add 1/10 volume NaAc (3M, PH5.2), the equal-volume Virahol ,-20 ℃ of precipitations 2 hours.4 ℃, 12000 rev/mins, centrifugal 15 minutes, wash once with 75% ethanol.Vacuum-drying is dissolved RNA again with no RNA enzyme deionized water.
The acquisition of 2 genome A fragment RF243 genes is carried out RT-PCR to the viral RNA that extracts, and uses random primer and carries out the RT reaction, and reaction system is 20 μ l, according to superscript
TMThe explanation of II reverse transcription reagent is operated.Design specific amplification RF243 PCR primer, primer sequence is: Paf:5`-gcggaattcgatgacgaacctgcaagatcaaac-3`, Par:5`-ccgaattccaaggtcctcatcagagacagcc-3`, with the RT product is template, adding 10 μ l goes in the PCR reaction system, the PCR reaction system is 100 μ l, other solution add according to Expand High Fidelity PCR System specification sheets, reaction conditions is as follows: pre-94 ℃ of 3min of sex change, loop parameter is 94 ℃ of 10sec, 55 ℃ of 20sec, 72 ℃ of 3min, circulate 30 times, 72 ℃ are extended 7min.
RNA obtains the fragment (Fig. 2) that length is 3.1Kb (SEQ ID NO:3) through RT-PCR amplification IBDV-Gx strain virus.This fragment comprises the complete great opening of IBDVA fragment gene and reads frame, gene sequencing result and the aminoacid sequence of deriving (SEQ ID NO:4) thereof.
Use magnificent Shun's sepharose and reclaim test kit purifying recovery PCR product, operation to specifications.The PCR product is connected with pMD18-T, the transformed into escherichia coli competent cell.The alkaline lysis method of extracting plasmid is identified with PCR, and PCR primers designed and condition are the same, identifies that correct recombinant plasmid delivers Dalian TaKaRa biotech firm order-checking.
3, the optimization transformation of VP2 gene reaches to synthesize the pichia spp codon is had a liking among the table input DNAstar software Editseq partially, to record sequence in the step 2 is prototype, carry out molecular modification and modification with software, the new gene order of acquisition (SEQ ID NO:1) is carried out synthetic.
4, the amplification of optimization back VP2 gene is a stencil design specific amplification VP2 gene primer with the optimized gene sequence, introduces XholI and XbaI enzyme cutting site at the primer two ends.Primer sequence is: Pvp2f:5`-gcgctcgagaagagagaggctgaagcaatgactaacttgc-3`, Pvp2r:5`-gcctctagaagcaccagcgatcttcaatgg-3`, gene with synthetic is that template is carried out pcr amplification, reaction system 100 μ l, template 2 μ l, other solution adds according to Expand High Fidelity PCR System specification sheets, reaction conditions is as follows: 94 ℃ of pre-sex change, 3min, loop parameter is 94 ℃ of 10sec, 55 ℃ of 20sec, 72 ℃ of 1min, circulate 30 times, 72 ℃ are extended 7min.
The structure and the evaluation of [embodiment 2] recombination yeast carrier
1, the structure of recombination yeast carrier will be optimized the VP2 gene and yeast vector is used XholI and XbaI enzyme cutting respectively, reclaim test kit with sepharose and reclaim.Carrier CIAP dephosphorylation.External source fragment and carrier T
4Dna ligase carries out ligation.Make up collection of illustrative plates and see Fig. 1.To connect product transformed into escherichia coli competent cell.
2, the extraction of recombinant plasmid and evaluation are pressed (screening pPICZ α VP2 plasmid microbiotic selection Zeocin among the activity adding less salt LB with corresponding microbiotic
TM), picking list colony inoculation LB.The alkaline lysis method of extracting plasmid.Identify with PCR method, PCR primers designed and condition are with embodiment 1, pcr amplification goes out 1.3Kb band (Fig. 3), to identify that correct plasmid delivers Dalian TaKaRa biotech firm order-checking, sequencing result shows that the gene of amplification meets the result of synthetic gene order-checking, gene inserts correct, conforms to the carrier reading frame.
[embodiment 3] conversion of recombination yeast carrier, the detection of restructuring yeast strains and screening
1, the transformed yeast competent cell is with recombination yeast carrier SacI linearization for enzyme restriction, and the transformed yeast competent cell transforms method of instruction according to Invitrogen company pichia spp electricity and carries out.To transform back yeast coating and contain antibiotic YPDS flat board (the recombination microzyme microbiotic selection Zeocin that screening pPICZ α VP2 transforms
TM).
2, the screening of yeast recon and 10 yeast lists of evaluation picking bacterium colony, inoculation 5ml contains antibiotic YPD, and incubated overnight is centrifugal, discards nutrient solution, with the washing of 5ml sterilization deionization once.Use Proteinase K, SDS digested overnight yeast, centrifugal, supernatant liquor is gone to another 1.5ml centrifuge tube, phenol, chloroform extracting 3 times, dehydrated alcohol precipitation.Use AOX1 5` and 3` primer and carry out PCR, the PCR condition is: pre-94 ℃ of 3min of sex change, and loop parameter is 94 ℃ of 1min, 55 ℃ of 1min, 72 ℃ of 1min, circulates 30 times, 72 ℃ are extended 7min.
Use Proteinase K-SDS digesting yeast bacterium, extract cerevisiae dna, carry out PCR with AOX1 5` and 3` primer, when exogenous genetic fragment is integrated into the yeast genes group, then should there be one in the PCR product than the dna fragmentation about the long 593bp of exogenous genetic fragment, otherwise has only the AOX gene fragment about a treaty 2.2Kb.The yeast phenotype is Mut when correct when external source fragment and yeast genes group are integrated
+, at this moment two dna fragmentations of exogenous genetic fragment+593bp and 2.2Kb can appear in the PCR product, integrate that the yeast phenotype is Mut when incorrect
s, at this moment have only exogenous genetic fragment+593bp dna fragmentation in the PCR product, see Fig. 4, by screening, obtain 545 strain recombination yeasts.
3, the small-scale abduction delivering of yeast recon identifies that with PCR phenotype is Mut
+Restructuring yeast strains inoculation 25ml BMGY, 30 ℃ of shake-flask culture (RPM300) are cultivated the nectar degree to OD
600Be that centrifugal (3000g 5min), with the 5ml BMMY yeast cell that suspends again, continued 30 ℃ of shake-flask culture (RPM300) in 8 o'clock.Add anhydrous methanol to 1% concentration in per 24 hours, draw bacterium liquid 100 μ l, centrifugal, supernatant is moved in another 1.5ml centrifuge tube, frozen in-70 ℃ of refrigerators.Collected whole bacterium liquid in 120 hours behind the access BMMY, centrifugal, supernatant is moved into another sterilising vessel, frozen in-70 ℃ of refrigerators.
4, the evaluation picking of expressing protein homophyletic yeast abduction delivering not, to induce the sample of results to get 50 μ l on a small scale, add 2 times of SDS sample-loading buffer 50 μ l, boiled 10 minutes, carry out SDS-PAGE electrophoresis, Western blot (with reference to " molecular cloning " second edition) mensuration expression amount, positive restructuring yeast strains secretory protein can be seen Fig. 5 with anti-IBDV monoclonal antibody reactive, same strain yeast different time expressing protein can access the amount of observing from 24 times hours, along with time lengthening, 72 hours protein concentration maximums.To express proteic expression amount be not different the identical time of homophyletic yeast, relies on SDS-PAGE to screen the high bacterial strain of expression amount and be optimized for engineering strain, and expression amount reaches 400mg/L.Western-blot result prove expressing protein can with specific antibody generation immune response.
The immunogenic mensuration of test example 1 recombinant protein of the present invention
Recombinant protein and the emulsification in 1: 1 of oily adjuvant that embodiment 2 is prepared divide 3 dosage by the protein content after the emulsification: 100 μ g/ml, 200 μ g/ml, 1000 μ g/ml.Get 4 age in week 50 of SPF chickens, divide 5 groups, 10 every group.1,2,3 groups are recombinant protein immune group of the present invention: 1 group of immunizing dose is 50 μ g/0.5ml/, and 2 groups of immunizing doses are 100 μ g/0.5ml/, and 3 groups of immunizing doses are 500 μ g/0.5ml/; 4 groups is conventional inactivated vaccine (the infectious bursal disease inactivated vaccine is available from biotechnology development company of dimension section of Harbin Veterinary Medicine Inst., China Academy of Agriculture) immune group, and immunizing dose is 0.5ml/; Immunization route is the chest muscle injection; 5 groups are no immune control group.The blood sampling in per 7 days of each group immunity back detects fine jade and expands antibody, and gets 5 in back 14 days each groups of immunity and attack poison, and remaining 5 of each group attack poison in immunity in the time of back 28 days.Attack the poison back and observed 7 days, the record death toll is calculated protection ratio.Test is carried out in negative pressure shield retaining (Australian import).
Immunity was attacked poison after 14 days, attacked poison back 7 days, and conventional deactivation vaccine immune group survival rate is 100%, and subunit vaccine immune group survival rate of the present invention is more than 80%, and to attack malicious control group survival rate be not 20% in immunity.Immunity was attacked poison after 28 days, attacked poison back 7 days, and conventional inactivated vaccine group survival rate is 100%, and subunit vaccine immune group survival rate of the present invention is 100%, and to attack malicious control group survival rate be not 20% in immunity.Attack malicious result and show,, can produce the immune protective efficiency that opposing virulent IBDV causes death and attacks with IBDV recombinant VP 2 protein immunization SPF chicken of the present invention.
The anti-gene engineering subunit vaccine of chicken's infectious bursal disease toxicology test of test example 2 the present invention
In order to investigate the toxicology reaction of test chicken after to the anti-gene engineering subunit vaccine of chicken's infectious bursal disease immunization of the present invention, this test adopt heavy dose of inoculation methods analyst the mutual relationship between vaccine and the body after the genetic engineering subunit vaccine inoculation of the present invention.
Concrete test is as follows:
Recombinant protein that embodiment 2 is prepared and oily adjuvant were in ratio emulsification in 1: 1, test chicken to 7 ages in days, 14 ages in days and 60 ages in days carries out the chest muscle injection, every chicken inoculation 1ml (being equivalent to 2 immunizing doses), the mental status and partial response situation that the chicken group is observed in the inoculation back.
As a result, tubercle all appears in the injection site of all test chickens, and the 7th day surrounding tissue in injection back forms granuloma, and absorption portion does not form packing.All test chickens all do not have other systemic reactions, and spiritual appetite is unaffected, grow normal.Test shows, promptly use 2 times of vaccine immunity dosage test injection chickens, can not cause the clinical response that test chicken is serious yet, the test-results explanation, protein subunit vaccine of the present invention is safe, both can be used for the immunity of above chicken in 5 ages in week, can be used for again 2 age in week chick immunity, even can implement immunization to the chicken in 1 age in week.
The safety testing result of table 1 vaccine of the present invention
The anti-gene engineering subunit vaccine of chicken's infectious bursal disease environment of test example 3 the present invention discharges safety evaluation
Year December in June, 2004 to 2004, (former Taiping District) Harbin, democracy township veterinary institute experimental animal feedlot has carried out environment release to the anti-gene engineering subunit vaccine of chicken's infectious bursal disease of the present invention in the Harbin City, Heilungkiang, and total experimental scale is 5000 plumage parts.
Concrete test method and result are as follows:
1 chicken infectivity bursa of Fabricius virus gene protein is in the intravital antibody growth and decline of chicken
With 50 μ g recombinant protein immunity test of the present invention chicken, extract 40 of chickens from vaccinated flock at random in 6 months after the vaccine inoculation, and from removing onesize sample as experimental control with the non-immune chicken of criticizing, blood sample collection, with the production of AG test detection chicken group serum antibody, the serology monitoring result shows that the immunity back began to occur antibody response in 1 week, tiring reaches 3.12 ± 0.45log2, and peaking in 8 weeks in immunity back, to tire be 5.31 ± 0.36log2.Every month antibody titer is subdued gradually later on, still can reach 4.07 ± 0.21log2 to 150 days antibody titers, and antibody titer can reach 3.38 ± 0.67log2 in 180 days.After the vaccine inoculation 6 months are per 30 days; extract 10 of chickens from vaccinated flock at random; and from getting onesize sample as experimental control with the non-vaccinated flock of criticizing; attack after the chicken group inoculates immanoprotection action after the subunit vaccine and comes across immunity 10 days with chicken infectivity bursa of Fabricius virus vvIBVDV-GX strain virulent.Immunity still can make the protection after the immune chicken of vaccinated flock 80% obtains the virulent attack in back 150 days.Immunity back different time antibody titer and chicken group are to the protection result (seeing Table 2) of strong virus attack.
Table 2 vaccine immunity extended period of the present invention test-results
2 passages are to the influence of recombinant pichia yeast strain stability
With restructuring yeast strains of the present invention continuous passage to 20 time, get the 5th, 10,15,20 generation bacterial strain carry out abduction delivering, expressing quantity does not have difference, with expressing protein phosphate buffered saline buffer (PBS, PH7.4) be diluted to 200 μ g/mL, recombinant protein solution mixed by 1: 1 with white oil sapn adjuvant put into the colloidal mill homogenizer then, make its emulsification, with the preparation subunit vaccine immunity test chicken, with 50 μ g/0.5ml/ plumages through chest muscle injection inoculation approach.Chicken infectivity bursa of Fabricius virus vvIBDV-GX strain virulent was attacked in immunity in back 28 days; each generation yeast expression protein Preparation vaccine all can reach the protection ratio more than 95%; protection test shows; the subunit vaccine that different generation restructuring yeast strains abduction delivering albumen are made does not change to the protection effect of immune chicken; can make the attack of immune chicken opposing chicken infectivity bursa of Fabricius virus vvIBVDV-GX strain virulent; the foreign gene that proof is inserted can be in zymic goes down to posterity process for a long time heredity stably and give expression to recombinant VP 2 albumen; expressing protein can stimulate the immune response of body generation at chicken infectivity bursa of Fabricius virus, and the chicken group uses recombiant vaccine not have the potential safety hazard (seeing Table 3) of immuning failure.
The different generation restructuring yeast strains of table 3 expressing protein is to the protection effect of chicken
3 express the transfer case of anti-infectious bursal disease subunit vaccine restructuring yeast strains to environment
After the off- test 1,2,3,4,8,12 weeks, the cleaning piece of the feed of acquisition test chicken house, drinking-water, dust, ight soil and cage tool is stored in 50% GBS, makes the homogenate of dilution in 1: 2 after the glass grinding device grinds, and inoculation contains Zeocin
TMIn the antibiotic YPD nutrient solution, wave and culture spends the night, and to medium centrifugal, is that Time Inc.'s yeast genes group is extracted test kit extraction cerevisiae dna with sky, Beijing, follows the tracks of detection with PCR method.PCR uses primer: pu5 '-tactattgccagcattgctgc-3, pr5 '-ggggacccgcgaacggat-3 '.Condition 94 ℃/3 minutes; 94 ℃/30 seconds, 56 ℃/30 seconds, 72 ℃/3 minutes and 30 seconds, 30 circulations; Last 72 ℃ were extended 10 minutes.PCR detected result (table 4) shows, can not be separated to recombination yeast after the off-test, and it is residual to illustrate that recombination microzyme of the present invention does not have in environment.
Restructuring yeast strains residual in environment after table 4 off-test
Evidence, the anti-gene engineering subunit vaccine of chicken's infectious bursal disease of the present invention is safe to the chicken group, and untoward reaction do not occur behind the immunization chicken immune; After the test chicken immunity, produce antibody rapidly, make body can resist the invasion and attack of the wild poison of chicken infectivity bursa of Fabricius virus; After the off-test, there be not the residual of transgenic microorganism in the environment.
In sum; can stimulate body to produce high titer antibody after the anti-gene engineering subunit vaccine of chicken's infectious bursal disease immunity of the present invention; the antibody extended period reached more than 6 months; make immune chicken obtain chicken infectivity bursa of Fabricius virus is protected for a long time; protection ratio is more than 95%, and duration of immunity can reach 3 months.Expressing the proteic restructuring yeast strains of chicken infectivity bursa of Fabricius virus can the stably express viral protein, does not have difference between its each generation of expressed proteins immunogenicity.Can not detect the existence of recombination microzyme after the off-test in the environment, prove that this tested object itself is as safe as a house, reliable to the chicken group, can not work the mischief environment.Therefore, the anti-gene engineering subunit vaccine of chicken's infectious bursal disease with yeast expression is the high vaccine of a kind of security.
SEQUENCE?LISTING
＜110〉Harbin Veterinary Medicine Inst., China Academy of Agriculture
＜120〉chicken infectivity bursa of Fabricius virus VP 2 cDNA, its expression vector, expressed recombinant protein and application
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gagacttcca?cttacaactt?gactgttggt?gacactggtt?ccggtttgat?cgttttcttc 180
ccaggtttcc?caggttccat?cgttggtgct?cactacactt?tgcaatccaa?cggtaactac 240
aagttcgacc?aaatgttgtt?gactgctcaa?aacttgccag?cttcctacaa?ctactgtaga 300
ttggtttcca?gatccttgac?tgttagatcc?tccactttgc?caggtggtgt?ttacgctttg 360
aacggtacta?tcaacgctgt?tactttccaa?ggttccttgt?ccgagttgac?tgacgtttcc 420
tacaacggtt?tgatgtccgc?tactgctaac?atcaacgaca?agatcggtaa?cgttttggtt 480
ggtgagggtg?ttactgtttt?gtccttgcca?acttcctacg?acttgggtta?cgttagattg 540
ggtgacccaa?tcccagctat?cggtttggac?ccaaagatgg?ttgctacttg?tgactcctcc 600
gacagaccaa?gagtttacac?tatcactgct?gctgacgact?accaattctc?ctcccaatac 660
caagctggtg?gtgttactat?cactttgttc?tccgctaaca?tcgacgctat?cacttccttg 720
tccatcggtg?gtgagttggt?tttccaaact?tccgttcaag?gtttgatctt?gggtgctact 780
atctacttga?tcggtttcga?cggtactgct?gttatcacta?gagctgttgc?tgctgacaac 840
ggtttgactg?ctggtactga?caacttgatg?ccattcaaca?tcgttatccc?aacttccgag 900
atcactcaac?caatcacttc?catcaagttg?gagatcgtta?cttccaagtc?cggtggtcaa 960
gctggtgacc?aaatgtcctg?gtccgcttcc?ggttccttgg?ctgttactat?ccacggtggt 1020
aactacccag?gtgctttgag?accagttact?ttggttgctt?acgagagagt?tgctactggt 1080
tccgttgtta?ctgttgctgg?tgtttccaac?ttcgagttga?tcccaaaccc?agagttggct 1140
aagaacttgg?ttactgagta?cggtagattc?gacccaggtg?ctatgaacta?cactaagttg 1200
atcttgtccg?agagagacag?attgggtatc?aagactgttt?ggccaactag?agagtacact 1260
gacttcagag?agtacttcat?ggaggttgct?gacttgaact?ccccattgaa?gatcgctggt 1320
gct 1323
<210>2
<211>3039
<212>DNA
<213>chicken?infectious?busal?disease?viru
<400>2
atgacgaacc?tgcaagatca?aacccaacag?attgttccgt?tcatacggag?ccttctgatg 60
ccaacaaccg?gaccggcgtc?cattccggac?gacaccctag?agaagcacac?tctcaggtca 120
gagacctcga?cctacaattt?gactgtgggg?gacacagggt?cagggctaat?tgtctttttc 180
cctggtttcc?ctggctcaat?tgtgggtgct?cactacacac?tgcagagcaa?tgggaactac 240
aagttcgatc?agatgctcct?gacggcccag?aacctaccgg?ccagctacaa?ctactgcagg 300
ctagtgagtc?ggagtcttac?agtgaggtca?agcacactcc?ctggtggcgt?ttatgcacta 360
aatggcacca?taaacgccgt?gaccttccaa?ggaagcctga?gtgaactgac?agatgttagc 420
tacaatgggt?tgatgtctgc?aacagccaac?atcaacgaca?aaatcgggaa?cgtcctagta 480
ggggaagggg?taaccgtcct?cagcttaccc?acatcatatg?atcttgggta?tgtgagactc 540
ggtgacccca?ttcccgctat?agggctcgac?ccaaaaatgg?tagcaacatg?tgacagcagt 600
gacaggccca?gagtctacac?cataactgca?gccgatgatt?accaattctc?atcacagtac 660
caagcaggtg?gggtaacaat?cacactgttc?tcagctaata?tcgatgccat?cacaagtctc 720
agcatcgggg?gagaactcgt?gtttcaaaca?agcgtccaag?gccttatact?gggtgctacc 780
atctacctta?taggctttga?tgggactgcg?gtaatcacca?gagctgtggc?cgcagacaat 840
gggctaacgg?ccggcactga?caaccttatg?ccattcaata?ttgtgattcc?aaccagcgag 900
ataacccagc?caatcacatc?catcaaactg?gagatagtaa?cctccaaaag?tggtggtcag 960
gcgggggacc?agatgtcatg?gtcagcaagt?gggagcctag?cagtgacgat?ccacggtggc 1020
aactatccag?gggccctccg?tcccgtcaca?ctagtagcct?acgaaagagt?ggcaacagga 1080
tctgtcgtta?cggtcgccgg?ggtgagcaac?ttcgagctga?tcccaaatcc?tgaactagca 1140
aagaacctgg?tcacagaata?cggccgattt?gacccaggag?ccatgaacta?cacaaaattg 1200
atactgagtg?agagggaccg?tcttggcatc?aagaccgtat?ggccaacaag?ggagtacact 1260
gactttcgcg?agtacttcat?ggaggtggcc?gacctcaact?ctcccctgaa?gattgcagga 1320
gcatttggct?tcaaagacat?aatccgggcc?ctaaggagga?tagctgtgcc?ggtggtatct 1380
acattgttcc?cacccgccgc?tcccctagcc?catgcaattg?gggaaggtgt?agactacctg 1440
ctgggcgatg?aggcacaggc?tgcttcagga?actgctcgag?ccgcgtcagg?aaaagcaaga 1500
gctgcctcag?gccgcataag?gcagctaact?ctcgccgccg?acaaggggta?cgaggtagtc 1560
gcgaatctgt?ttcaggtgcc?ccagaatcct?gtagtcgacg?ggattctcgc?ttcacctggg 1620
atactccgcg?gtgcacacaa?cctcgactgc?gtgttgagag?agggtgccac?gctattccct 1680
gtggtcatca?cgacagtgga?agatgccatg?acacccaaag?cactgaacag?caaaatgttt 1740
gctgtcattg?aaggcgtgcg?agaagatctc?caacctccat?ctcaaagagg?atccttcata 1800
cgaactctct?ccggacatag?agtctatgga?tatgctccag?atggggtact?tccactggag 1860
actgggagag?attacaccgt?ggtcccaata?gatgatgtct?gggacgacag?cattatgctg 1920
tccaaagacc?ccatacctcc?tattgtggga?aacagtggaa?acctagccat?agcttacatg 1980
gatgtgtttc?gacccaaagt?ccccatccat?gtggccatga?cgggagccct?caacgcctat 2040
ggcgagattg?agaacgtgag?ctttagaagc?accaagctcg?ccactgcaca?ccgacttggc 2100
ctcaagttgg?ctggtcccgg?tgcatttgac?gtgaacaccg?ggtccaactg?ggcgacgttt 2160
atcaaacgtt?ttcctcacaa?tccacgcgac?tgggacaggc?tcccttacct?caaccttcca 2220
taccttccac?ccaatgcagg?acgccagtac?gacctggcta?tggccgcttc?agagttcaaa 2280
gaaacccccg?aactcgagag?cgccgtcaga?gccatggaag?cagcagccga?cgtggaccca 2340
ctgttccatt?ctgcgctcag?tgtgttcatg?tggctggaag?aaaatgggat?tgtgaccgac 2400
atggccaact?tcgcactcag?cgacccgaac?gcccatcgga?tgcgcaattt?tctcgcaaac 2460
gcaccacaag?caggcagcaa?gtcgcaaaga?gccaagtacg?ggacagcagg?ctacggagtg 2520
gaggcccggg?gccccactcc?agaggaagca?cagagggaaa?aagacacacg?gatctcaaag 2580
aagatggaga?ctatgggcat?ctactttgca?acaccagaat?gggtagcact?caatgggcac 2640
cgggggccaa?gccccggcca?gctgaagtac?tggcagaaca?cacgagaaat?acctgatcca 2700
aacgaggact?acctagacta?cgtgcatgca?gagaagagcc?ggttggcatc?agaagaacaa 2760
atcctaaggg?cagctacgtc?gatctacggg?gctccaggac?aggcagagcc?accccaagcc 2820
ttcatagacg?aagtcgccaa?agtctatgaa?atcaaccatg?ggcgtggccc?caaccaagaa 2880
cagatgaaag?atctgctctt?gactgcgatg?gagatgaagc?atcgcaatcc?caggcgggct 2940
ccaccaaagc?ccaagccaaa?acccaatgtt?ccaacacaga?gaccccctgg?tcggctgggc 3000
cgctggatca?gggctgtctc?tgatgaggac?cttgagtga 3039
<210>3
<211>1012
<212>PRT
<213>chicken?infectious?busal?disease?virus
<400>3
Met?Thr?Asn?Leu?Gln?Asp?Gln?Thr?Gln?Gln?Ile?Val?Pro?Phe?Ile?Arg
1 5 10 15
Ser?Leu?Leu?Met?Pro?Thr?Thr?Gly?Pro?Ala?Ser?Ile?Pro?Asp?Asp?Thr
20 25 30
Leu?Glu?Lys?His?Thr?Leu?Arg?Ser?Glu?Thr?Ser?Thr?Tyr?Asn?Leu?Thr
35 40 45
Val?Gly?Asp?Thr?Gly?Ser?Gly?Leu?Ile?Val?Phe?Phe?Pro?Gly?Phe?Pro
50 55 60
Gly?Ser?Ile?Val?Gly?Ala?His?Tyr?Thr?Leu?Gln?Ser?Asn?Gly?Asn?Tyr
65 70 75 80
Lys?Phe?Asp?Gln?Met?Leu?Leu?Thr?Ala?Gln?Asn?Leu?Pro?Ala?Ser?Tyr
85 90 95
Asn?Tyr?Cys?Arg?Leu?Val?Ser?Arg?Ser?Leu?Thr?Val?Arg?Ser?Ser?Thr
100 105 110
Leu?Pro?Gly?Gly?Val?Tyr?Ala?Leu?Asn?Gly?Thr?Ile?Asn?Ala?Val?Thr
115 120 125
Phe?Gln?Gly?Ser?Leu?Ser?Glu?Leu?Thr?Asp?Val?Ser?Tyr?Asn?GIy?Leu
130 135 140
Met?Ser?Ala?Thr?Ala?Asn?Ile?Asn?Asp?Lys?Ile?Gly?Asn?Val?Leu?Val
145 150 155 160
Gly?Glu?Gly?Val?Thr?Val?Leu?Ser?Leu?Pro?Thr?Ser?Tyr?Asp?Leu?Gly
165 170 175
Tyr?Val?Arg?Leu?Gly?Asp?Pro?Ile?Pro?Ala?Ile?Gly?Leu?Asp?Pro?Lys
180 185 190
Met?Val?Ala?Thr?Cys?Asp?Ser?Ser?Asp?Arg?Pro?Arg?Val?Tyr?Thr?Ile
195 200 205
Thr?Ala?Ala?Asp?Asp?Tyr?Gln?Phe?Ser?Ser?Gln?Tyr?Gln?Ala?Gly?Gly
210 215 220
Val?Thr?Ile?Thr?Leu?Phe?Ser?Ala?Asn?Ile?Asp?Ala?Ile?Thr?Ser?Leu
225 230 235 240
Ser?Ile?Gly?Gly?Glu?Leu?Val?Phe?Gln?Thr?Ser?Val?Gln?Gly?Leu?Ile
245 250 255
Leu?Gly?Ala?Thr?Ile?Tyr?Leu?Ile?Gly?Phe?Asp?Gly?Thr?Ala?Val?Ile
260 265 270
Thr?Arg?Ala?Val?Ala?Ala?Asp?Asn?Gly?Leu?Thr?Ala?Gly?Thr?Asp?Asn
275 280 285
Leu?Met?Pro?Phe?Asn?Ile?Val?Ile?Pro?Thr?Ser?Glu?Ile?Thr?Gln?Pro
290 295 300
Ile?Thr?Ser?Ile?Lys?Leu?Glu?Ile?Val?Thr?Ser?Lys?Ser?Gly?Gly?Gln
305 310 315 320
Ala?Gly?Asp?Gln?Met?Ser?Trp?Ser?Ala?Ser?Gly?Ser?Leu?Ala?Val?Thr
325 330 335
Ile?His?Gly?Gly?Asn?Tyr?Pro?Gly?Ala?Leu?Arg?Pro?Val?Thr?Leu?Val
340 345 350
Ala?Tyr?Glu?Arg?Val?Ala?Thr?Gly?Ser?Val?Val?Thr?Val?Ala?Gly?Val
355 360 365
Ser?Asn?Phe?Glu?Leu?Ile?Pro?Asn?Pro?Glu?Leu?Ala?Lys?Asn?Leu?Val
370 375 380
Thr?Glu?Tyr?Gly?Arg?Phe?Asp?Pro?Gly?Ala?Met?Asn?Tyr?Thr?Lys?Leu
385 390 395 400
Ile?Leu?Ser?Glu?Arg?Asp?Arg?Leu?Gly?Ile?Lys?Thr?Val?Trp?Pro?Thr
405 410 415
Arg?Glu?Tyr?Thr?Asp?Phe?Arg?Glu?Tyr?Phe?Met?Glu?Val?Ala?Asp?Leu
420 425 430
Asn?Ser?Pro?Leu?Lys?Ile?Ala?Gly?Ala?Phe?Gly?Phe?Lys?Asp?Ile?Ile
435 440 445
Arg?Ala?Leu?Arg?Arg?Ile?Ala?Val?Pro?Val?Val?Ser?Thr?Leu?Phe?Pro
450 455 460
Pro?Ala?Ala?Pro?Leu?Ala?His?Ala?Ile?Gly?Glu?Gly?Val?Asp?Tyr?Leu
465 470 475 480
Leu?Gly?Asp?Glu?Ala?Gln?Ala?Ala?Ser?Gly?Thr?Ala?Arg?Ala?Ala?Ser
485 490 495
Gly?Lys?Ala?Arg?Ala?Ala?Ser?Gly?Arg?Ile?Arg?Gln?Leu?Thr?Leu?Ala
500 505 510
Ala?Asp?Lys?Gly?Tyr?Glu?Val?Val?Ala?Asn?Leu?Phe?Gln?Val?Pro?Gln
515 520 525
Asn?Pro?Val?Val?Asp?Gly?Ile?Leu?Ala?Ser?Pro?Gly?Ile?Leu?Arg?Gly
530 535 540
Ala?His?Asn?Leu?Asp?Cys?Val?Leu?Arg?Glu?Gly?Ala?Thr?Leu?Phe?Pro
545 550 555 560
Val?Val?Ile?Thr?Thr?Val?Glu?Asp?Ala?Met?Thr?Pro?Lys?Ala?Leu?Asn
565 570 575
Ser?Lys?Met?Phe?Ala?Val?Ile?Glu?Gly?Val?Arg?GIu?Asp?Leu?Gln?Pro
580 585 590
Pro?Ser?Gln?Arg?Gly?Ser?Phe?Ile?Arg?Thr?Leu?Ser?Gly?His?Arg?Val
595 600 605
Tyr?Gly?Tyr?Ala?Pro?Asp?Gly?Val?Leu?Pro?Leu?Glu?Thr?Gly?Arg?Asp
610 615 620
Tyr?Thr?Val?Val?Pro?Ile?Asp?Asp?Val?Trp?Asp?Asp?Ser?Ile?Met?Leu
625 630 635 640
Ser?Lys?Asp?Pro?Ile?Pro?Pro?Ile?Val?Gly?Asn?Ser?Gly?Asn?Leu?Ala
645 650 655
Ile?Ala?Tyr?Met?Asp?Val?Phe?Arg?Pro?Lys?Val?Pro?Ile?His?Val?Ala
660 665 670
Met?Thr?Gly?Ala?Leu?Asn?Ala?Tyr?Gly?Glu?Ile?Glu?Asn?Val?Ser?Phe
675 680 685
Arg?Ser?Thr?Lys?Leu?Ala?Thr?Ala?His?Arg?Leu?Gly?Leu?Lys?Leu?Ala
690 695 700
Gly?Pro?Gly?Ala?Phe?Asp?Val?Asn?Thr?Gly?Ser?Asn?Trp?Ala?Thr?Phe
705 710 715 720
Ile?Lys?Arg?Phe?Pro?His?Asn?Pro?Arg?Asp?Trp?Asp?Arg?Leu?Pro?Tyr
725 730 735
Leu?Asn?Leu?Pro?Tyr?Leu?Pro?Pro?Asn?Ala?Gly?Arg?Gln?Tyr?Asp?Leu
740 745 750
Ala?Met?Ala?Ala?Ser?Glu?Phe?Lys?Glu?Thr?Pro?Glu?Leu?Glu?Ser?Ala
755 760 765
Val?Arg?Ala?Met?Glu?Ala?Ala?Ala?Asn?Val?Asp?Pro?Leu?Phe?His?Ser
770 775 780
Ala?Leu?Ser?Val?Phe?Met?Trp?Leu?Glu?Glu?Asn?Gly?Ile?Val?Thr?Asp
785 790 795 800
Met?Ala?Asn?Phe?Ala?Leu?Ser?Asp?Pro?Asn?Ala?His?Arg?Met?Arg?Asn
805 810 815
Phe?Leu?Ala?Asn?Ala?Pro?Gln?Ala?Gly?Ser?Lys?Ser?Gln?Arg?Ala?Lys
820 825 830
Tyr?Gly?Thr?Ala?Gly?Tyr?Gly?Val?Glu?Ala?Arg?Gly?Pro?Thr?Pro?Glu
835 840 845
Glu?Ala?Gln?Arg?Glu?Lys?Asp?Thr?Arg?Ile?Ser?Lys?Lys?Met?Glu?Thr
850 855 860
Met?Gly?Ile?Tyr?Phe?Ala?Thr?Pro?Glu?Trp?Val?Ala?Leu?Asn?Gly?His
865 870 875 880
Arg?Gly?Pro?Ser?Pro?Gly?Gln?Leu?Lys?Tyr?Trp?Gln?Asn?Thr?Arg?Glu
885 890 895
Ile?Pro?Asp?Pro?Asn?Glu?Asp?Tyr?Leu?Asp?Tyr?Val?His?Ala?Glu?Lys
900 905 910
Ser?Arg?Leu?Ala?Ser?Glu?Glu?Gln?Ile?Leu?Arg?Ala?Ala?Thr?Ser?Ile
915 920 925
Tyr?Gly?Ala?Pro?Gly?Gln?Ala?Glu?Pro?Pro?Gln?Ala?Phe?Ile?Asp?Glu
930 935 940
Val?Ala?Lys?Val?Tyr?Glu?Ile?Asn?His?Gly?Arg?Gly?Pro?Asn?Gln?Glu
945 950 955 960
Gln?Met?Lys?Asp?Leu?Leu?Leu?Thr?Ala?Met?Glu?Met?Lys?His?Arg?Asn
965 970 975
Pro?Arg?Arg?Ala?Pro?Pro?Lys?Pro?Lys?Pro?Lys?Pro?Asn?Val?Pro?Thr
980 985 990
Gln?Arg?Pro?Pro?Gly?Arg?Leu?Gly?Arg?Trp?Ile?Arg?Ala?Val?Ser?Asp
995 1000 1005
Glu?Asp?Leu?Glu
1010
Claims (10)
1. the chicken infectivity bursa of Fabricius virus VP 2 cDNA of a transformation is characterized in that being the nucleotide sequence shown in the SEQ IDNO:1.
2. the recombinant yeast expression vector that contains claim 1 chicken infectivity bursa of Fabricius virus VP 2 cDNA.
3. recombinant yeast expression vector transformed host cells with claim 2.
4. a chicken infectivity bursa of Fabricius virus recombinant VP 2 albumen is characterized in that by the nucleotide sequence shown in the SEQ ID NO:1 of claim 1 coded.
5. one kind prepares the proteic method of claim 4 chicken infectivity bursa of Fabricius virus recombinant VP 2, may further comprise the steps:
With the recombinant yeast expression vector transformed yeast cell of claim 2, induce the proteic expression of chicken infectivity bursa of Fabricius virus recombinant VP 2, reclaim and the expressed chicken infectivity bursa of Fabricius virus recombinant VP 2 albumen of purifying.
6. according to the method for claim 5, it is characterized in that described yeast cell is a SMD1168 pichia spp cell.
7. the protein subunit recombinant vaccine of an anti-infectious bursal disease is characterized in that this vaccine contains the chicken infectivity bursa of Fabricius virus recombinant VP 2 albumen of the claim 4 of significant quantity.
8. according to the protein subunit recombinant vaccine of claim 7, it is characterized in that this vaccine also contains pharmaceutically acceptable adjuvant, carrier or excipient.
9. the purposes of the chicken infectivity bursa of Fabricius virus VP 2 cDNA of claim 1 in preparation control or diagnosis infectious bursal disease medicine.
10. the purposes of the chicken infectivity bursa of Fabricius virus recombinant VP 2 albumen of claim 4 in preparation control or diagnosis infectious bursal disease medicine.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200510135583 CN100475963C (en) | 2005-12-30 | 2005-12-30 | Chicken infectivity bursa of Fabricius virus VP2 cDNA, its expression vector, expressed recombinant protein and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
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| CN 200510135583 CN100475963C (en) | 2005-12-30 | 2005-12-30 | Chicken infectivity bursa of Fabricius virus VP2 cDNA, its expression vector, expressed recombinant protein and application thereof |
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| CN100475963C true CN100475963C (en) | 2009-04-08 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102277374B (en) * | 2011-07-27 | 2013-04-17 | 浙江诺倍威生物技术有限公司 | Preparation and application of genetic engineering subunit vaccine for infectious bursal disease |
| CN103849631B (en) * | 2012-11-28 | 2016-09-07 | 普莱柯生物工程股份有限公司 | Vaccine combination containing chicken infectivity bursa of Fabricius virus subunit antigen and its preparation method and application |
| CN102973935A (en) * | 2012-12-03 | 2013-03-20 | 青岛康地恩药业股份有限公司 | Chicken subunit triple vaccine, as well as preparation and application thereof |
| CN104761624B (en) * | 2015-04-23 | 2019-01-25 | 中国农业科学院生物技术研究所 | The preparation method and its product of chicken infectivity bursa of Fabricius virus antigen |
| CN105755015B (en) * | 2016-03-21 | 2020-10-27 | 中国农业科学院哈尔滨兽医研究所 | Recombinant yeast strain for expressing infectious bursal disease virus-like particles, protein expressed by recombinant yeast strain and application of recombinant yeast strain |
| CN107245105B (en) * | 2017-06-29 | 2021-01-12 | 河南科技大学 | HN-VP233-221aa fusion protein and preparation method and application thereof |
| CN107446029A (en) * | 2017-08-07 | 2017-12-08 | 新疆畜牧科学院 | A kind of Caprine arthritis encephalitis virus serum antibody diagnostic flag albumen p28 and preparation method thereof |
| CN109200281A (en) * | 2018-10-24 | 2019-01-15 | 山东农业大学 | The subunit vaccine of single stranded circle DNA virus GyV3 and its preparation |
| CN110872578B (en) * | 2019-11-29 | 2022-09-16 | 扬州优邦生物药品有限公司 | Variant infectious bursal disease virus, subunit vaccine, preparation method and application thereof |
| CN113493509B (en) * | 2021-07-23 | 2023-04-28 | 东北农业大学 | Tetravalent high-affinity antibody for resisting infectious bursal disease virus, and preparation and application thereof |
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Non-Patent Citations (3)
| Title |
|---|
| . Genbank,Vol.L42284 . 1998 |
| . Genbank,Vol.L42284 . 1998;传染性法氏囊病毒VP2在酵母细胞内的高效表达及其免疫原性研究. 王笑梅等.中国农业科学，第36卷第4期. 2003 * |
| 传染性法氏囊病毒VP2在酵母细胞内的高效表达及其免疫原性研究. 王笑梅等.中国农业科学，第36卷第4期. 2003 |
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