CN102277374B - Preparation and application of genetic engineering subunit vaccine for infectious bursal disease - Google Patents
Preparation and application of genetic engineering subunit vaccine for infectious bursal disease Download PDFInfo
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Abstract
The invention relates to preparation and application of a genetic engineering subunit vaccine for the infectious bursal disease. The inventor optimizes the recombinant expression method of VP2 protein of the infectious bursal disease virus, so that the expression yield of the VP2 protein is effectively increased, and the consistency of the expression product and natural protein is ensured. The method provided by the invention has simple process and low cost, and the subunit vaccine of the VP2 protein of the infectious bursal disease virus, which can be used safely, can be efficiently and stably obtained.
Description
Technical field
The invention belongs to biotechnology and field of immunology; More specifically, the present invention relates to a kind of preparation and application of gene engineering subunit vaccine of chicken's infectious bursal disease.
Background technology
Infectious bursal disease (Infectious bursal disease, IBD) be a kind of by infectious bursal disease virus (Infectious bursal disease virus, IBDV) acute, the height contagious disease that cause, often cause chicken group's mortality, cause simultaneously immunosuppression, so that chicken group vaccine inoculation immuning failure and secondary, complication roll up, cause serious financial loss.This disease has become serious, the most thorny transmissible disease of harm China's poultry husbandry, does not have at present special effect medicine therapeutic.
Means of prevention commonly used is vaccine inoculation at present, comprise attenuated vaccine and inactivated vaccine, but find so far from IBDV, new variant, virulent strain, highly virulent strain constantly occur, the change of molecular structure causes the change of viral virulence and the change that the host replys vaccine, it is popular so that traditional vaccine can not have been controlled, brings new difficulty and challenge for the prevention of disease and treatment.In recent years; China's poultry husbandry development rapidly; the particularly raising of the production level that increases, raises chickens of feeding chicken in largely scale field; the demand of annual bursal disease vaccine also increases rapidly; therefore be badly in need of developing a kind of safely and effectively product, can large-scale production and application under low-cost condition, prevent with utmost dispatch; the propagation of control virus, thereby the sound development of guarantee aquaculture.
Along with the development of Protocols in Molecular Biology, the research of virogene structure and function to be goed deep into, infectivity bursal disease virogene restructuring development reaches its maturity, and recombinant vaccine has been the main direction of development.Comprehensive relatively range gene engineered vaccine wherein has more unique advantage with protein subunit vaccine especially.Protein subunit vaccine never contains the malicious composition of living, and production and use procedure have been eliminated the possibility of polluting without loose poison possibility, is the worth safe vaccine of actively promoting the use of.
Formerly patent " 200410080065.8, a kind of preparation method and application of genetic engineering subunit vaccine of infectious bursal disease " in, subunit vaccine can stimulate body to produce neutralizing antibody and effectively support antiviral attack, but prokaryotic expression system need to carry out to cell precipitation numerous and diverse programs such as thawing ultrasonication, and have the residual endotoxic danger of possibility in the bacterial body, be restricted aspect the security of technological process of production simplification and vaccine.
Formerly in the patent " 200510135583.X, chicken infectivity bursa of Fabricius virus VP 2 cDNA, its expression vector, expressed recombinant protein and application ", goal gene VP2 has been carried out artificial reconstructed, complete genome sequence in yeast, expressed after synthetic.But the residual one section sequence that has on the carrier of target protein C end of expressing in this patent does not have natural sex, and has affected its immunogenicity.
Therefore, still need to improve the technology of preparing of gene engineering subunit vaccine of chicken's infectious bursal disease, to improve expression amount, the vaccine that adaptive immune originality is strong.
Summary of the invention
The object of the present invention is to provide preparation and the application of a kind of infectious bursal disease gene engineered subunit (VP2) vaccine.
In a first aspect of the present invention, the method for a kind of recombinant expressed Infectious bursal disease virus VP2 subunit is provided, the method comprises:
(1) provides the VP2 protein subunit encoding gene of transformation; Wherein, the cutting sequence of adding Proteinase K EX2 before 5 ' end initiator codon ATG of described VP2 protein subunit encoding gene is 3 ' end interpolation termination codon subsequence of described VP2 protein subunit encoding gene;
(2) (1) described VP2 protein subunit encoding gene is inserted into the α of Yeast expression carrier pPICZ α A-factor signal coding sequence downstream, obtains recombinant expression vector;
(3) recombinant expression vector with (2) transforms pichia spp, obtains the Pichia pastoris of restructuring;
(4) Pichia pastoris of the restructuring of (3) is carried out recombinant expressed, from express supernatant, obtain Infectious bursal disease virus VP2 subunit.
In another preference, in the step (1), also before 5 ' end of the cutting sequence of described Proteinase K EX2, add XhoI restriction enzyme site sequence; 3 ' end described termination codon subsequence adds NotI restriction enzyme site sequence.
In another preference, in the step (2), described VP2 protein subunit encoding gene is inserted between the XhoI/NotI restriction enzyme site of Yeast expression carrier pPICZ α A.
In another preference, the sequence of the VP2 protein subunit encoding gene of described transformation is shown in SEQ IDNO:1.
In another preference, described pichia spp is the X-33 bacterial strain.
In another preference, the recombinant expression vector electricity is transformed pichia spp, electric conversion condition is: 0.2cm electricity revolving cup, voltage 2.0KV, electric shock time 5ms, plasmid concentration is 7-10ug/10 μ L, competent cell 100 μ L; Add fast 1mL 1M Sorbitol Powder after electric the conversion, recovered 1 hour for 30 ℃.
In another preference, in the step (4), expression condition is: adopt inducing culture BMMY, the substratum pH value is 6.5 ± 0.5, and inducing temperature is 28.5 ± 1 ℃, and the methyl alcohol final concentration is 0.5 ± 0.2% (v/v).
In another preference, adopt methyl alcohol to carry out abduction delivering.Preferably, the final concentration of methyl alcohol is 0.5 ± 0.2% (v/v); More preferably final concentration is 0.5 ± 0.1% (v/v).
In another aspect of this invention, provide a kind of method for preparing the infectivity bursa of Fabricius virus VP 2 protein subunit vaccine, the method comprises:
Obtain Infectious bursal disease virus VP2 subunit with described method, the step of carrying out emulsification obtains the infectivity bursa of Fabricius virus VP 2 protein subunit vaccine.
In another preference, with described Infectious bursal disease virus VP2 subunit originally mix with white oil department, emulsification.
In another aspect of this invention, provide a kind of polynucleotide for recombinant expressed Infectious bursal disease virus VP2 subunit, its nucleotide sequence is shown in SEQ ID NO:1.
In another aspect of this invention, provide a kind of primer be used to obtaining described polynucleotide, the nucleotide sequence of described primer is shown in SEQ ID NO:2 and SEQ ID NO:3.
Other side of the present invention is because the disclosure of this paper is apparent to those skilled in the art.
Description of drawings
The cDNA sequence of Fig. 1, the improved VP2 gene of the present invention.
Fig. 2, the evaluation of recombinant expression plasmid double digestion, except rightmost side swimming lane is DNA Marker, other swimming lane is with XhoI/NotI and carries out the electrophoresis result that enzyme is cut.
Fig. 3, recombinant expression plasmid PCR identify, identifies with the electrophoresis of the amplified production that obtains behind 5 ' AOX and 3 ' the AOX primer amplification.
Fig. 4, utilize the goal gene Auele Specific Primer to carry out the result that recombinant expression plasmid PCR identifies.
Fig. 5, restructuring yeast strains PCR identify positive colony and phenotype thereof.
Fig. 6, recombinant VP 2 protein SDS-PAGE are identified.
Fig. 7, recombinant VP 2 protein immunoblot are identified (Western-Blot).
Fig. 8, indirect elisa method are measured the measurement result of neutralizing antibody level.
Embodiment
The inventor has optimized the recombinant expression method of Infectious bursal disease virus VP2 through deep research, Effective Raise VP2 protein expression output, guaranteed the consistence of expression product and native protein.Method technique of the present invention is simple, with low cost, can obtain efficiently and stably the Infectious bursal disease virus VP2 subunit vaccine that can use safely.
Expression system
In the past, people more adopted prokaryotic expression system to express VP2 albumen, and the expression process is complicated, difficult operation, and protein yield is difficult to improve.Therefore, the inventor is devoted to develop new VP2 protein expression system, to raising output, and the good albumen of adaptive immune originality, thereby can directly apply to the preparation vaccine.Through the comparison of multiple expression system, the inventor has finally selected Bichi yeast system.
Compare the characteristic of prokaryotic cell prokaryocyte and these two kinds of host cells of yeast, the advantage of yeast is very obvious.The pichia methanolica expression system can be translated accurately post-treatment with the recombinant protein of expressing and modify, close to native protein.The secretor type Yeast expression carrier can be secreted into target protein in the fermented supernatant fluid, and the background albumen major part of yeast self is all stayed in the cell precipitation, has removed numerous and diverse subsequent disposal such as precipitation fragmentation and purifying from.The fermented supernatant fluid that method of the present invention obtains can not done any processing and be directly used in the making subunit vaccine, and the result shows that this protein subunit vaccine can stimulate chicken to produce neutralizing antibody effectively.With respect to prokaryotic system expression products such as bacteriums, the vaccine that using yeast is produced is without any side effects, and technique is simple, and is with low cost, is fit to very much large-scale industrial production.
The present invention has carried out transformation and selection to this multiple yeast strain, according to the height of transformant growing state and expressing quantity, selects only yeast strain.The bacterial strain that pichia spp is commonly used has KM71H, GS115, X-33 etc., all is methyl alcohol nutritional type yeast, and at genome type, foreign gene has different integration and recombination form when inserting, and difference is arranged on the expression effect.As optimal way of the present invention, adopt X-33 as recombinant expressed Host Strains.
Genetic modification
Those skilled in the art all understand, and the key of utilizing recombination and expression techniques to prepare vaccine is that albumen and the native protein expressed have approaching antigenicity, also is that recombinant protein preferably possesses " natural sex ".With this end in view, the inventor has carried out the sequence improvement to VP2 protein expression construction.
α on the Yeast expression carrier pPICZ α A-factor signal peptide comprises one section spacer peptide that is comprised of the Lys-Arg-Glu-Ala-Glu-Ala aminoacid sequence of 83 amino acid whose leading peptides and back, the ripe signal peptide sequence processing of α-factor in two steps, embrane-associated protein enzyme KEX2 was to cut between Arg and the Glu in the asterisk position shown in the Lys-Arg*Glu-Ala-Glu-Ala before this, then proteolytic enzyme STE13 cuts Glu-Ala tumor-necrosis factor glycoproteins, i.e. position shown in the asterisk among the sequence Glu-Ala*Glu-Ala*.Leading peptide and spacer peptide are to the correct cutting of albumen and secrete most important, but sometimes because the cutting vigor of above-mentioned two kinds of gene products is different, can produce different cleavage sites at the N of foreign protein end, in many situations, the efficient of Stel13 cutting Glu-Ala tumor-necrosis factor glycoproteins is not high, make some albumen can hold residual (Glu-Ala) 1-2 fusion sequence that has at N in secretion, namely produced so-called signal peptide of yeast and cut incomplete phenomenon, this may affect the activity of foreign protein.
Therefore, in order to obtain to have the target protein of natural radioactivity, the inventor has done following design: one, four aminoacid sequences (Leu-Glu-Lys-Arg) in α-factor signal peptide downstream on the Yeast expression carrier pPICZ α A have been merged at the VP2 albumen n end, the first two aminoacid sequence is the restriction enzyme site of restriction enzyme XhoI, latter two aminoacid sequence is the cutting sequence of embrane-associated protein enzyme KEX2, namely, VP2 albumen has only merged the cutting sequence A AA-AGA (Lys-Arg) of one of them Proteinase K EX2 among the present invention, the purpose of design is the incomplete cutting of having avoided STE13 to cause like this, guarantee that the VP2 albumen of expressing can cut down fully from signal peptide, can residual unnecessary amino acid and affect its antigenicity, keep integrity and the natural radioactivity of albumen; Its two, add terminator TAA at VP2 gene 3 ' end, thereby removed two sections sequence c-myc epitope and polyhistidine tag on the carrier, same so that the target protein of expressing can be not residual except other the unnecessary sequences self.
Make a general survey of prior art, having no report has similar sequence transformation, so genetic modification of the present invention has unique novelty.The expression amount of fermented supernatant fluid target protein is up to 0.766mg/ml among the present invention, is the high expression level amount of utilizing at present that Yeast system carries out that recombinant vaccine institute obtains.Why obtain the high expression level amount, with above-mentioned gene order transformation direct relation is arranged.
Expression method
The method of recombinant expressed Infectious bursal disease virus VP2 of the present invention subunit mainly comprises:
(1) provides the VP2 protein subunit encoding gene of transformation;
(2) (1) described VP2 protein subunit encoding gene is inserted into the α of Yeast expression carrier pPICZ α A-factor signal coding sequence downstream, obtains recombinant expression vector;
(3) recombinant expression vector with (2) transforms pichia spp, obtains the Pichia pastoris of restructuring;
(4) Pichia pastoris of the restructuring of (3) is carried out recombinant expressed, from express supernatant, obtain Infectious bursal disease virus VP2 subunit.
As optimal way of the present invention, methyl alcohol is used to the expression of inducible protein.Preferably, the final concentration of methyl alcohol is 0.5 ± 0.2% (v/v); More preferably final concentration is 0.5 ± 0.1% (v/v).
The setting of voltage when turning because of electricity, electricity turns the concentration of time and plasmid, and the competent cell state is all influential to electric changing effect, so the inventor has also groped the electroporation parameter of electric conversion, selects suitable plasmid concentration and competent cell concentration.Grope through many experiments, the inventor has obtained better electricity and turned parameter: plasmid concentration is 7-10ug/10 μ L, competent cell 100 μ L; Add fast lmL 1M Sorbitol Powder after electric the conversion, recovered 1 hour for 30 ℃.In when operation, the last amount that adds 1M sorbitol of control so that cell concn a little some thickness for well.In addition, at the 1M sorbitol that adds 0 ℃ of 1mL, 30 ℃ left standstill recovery after 1 hour, added 1mL YPD liquid nutrient medium again, 30 ℃, 200rpm vibration recovery 1 hour, like this electricity turn effect can be better.
The optimization that turns condition to power on has solved generation false positive clone, and positive colony is few, and plasmid stability is low, the low problem that more often occurs when transforming that waits of copy number.
According to the characteristic of pichia spp, can carry out at normal temperatures protein expression, preferably, under 30 ± 5 ℃ temperature, express.
In an embodiment of the present invention; the inventor isolates highly virulent strain (vvIBDV) from the numerous epidemic strains of domestic infectious bursal disease (IBV); use the RT-PCR method; amplification obtains the open encoder block of IBDV main protection antigen VP2 gene; and with the VP2 gene clone to yeast expression vector pPICZ α A; obtain recombinant expression plasmid pPICZ α A-VP2; then this recombinant expression plasmid is changed in the pichia pastoris X-33 competent cell with electric method for transformation; identify by PCR; phenotypic screen and the screening of antibiotic concentration gradient obtain the positive restructuring yeast strains X-33-pPICZ α A-VP2 of high copy.The test of shaking flask abduction delivering obtains the Yeast engineering bacteria that stability and high efficiency is expressed, through agar diffusion test (ADIT), polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting (western-blot) identify and show, recombinant VP 2 albumen has biological activity and the immunogenicity of chicken infectivity bursa of Fabricius virus native protein.
The vaccine preparation
Adopt the VP2 albumen of the recombinant expressed acquisition of aforesaid method of the present invention, can be for the preparation of vaccine behind the process purifying; Perhaps culture supernatant (fermentation supernatant) is not done any processing and be directly used in the preparation vaccine.
The method for preparing vaccine is that those skilled in the art understand.Can adopt multiple preparation method or select multiple adjuvant, these are all in the present invention involved.
In an embodiment of the present invention, make oily adjuvant subunit vaccine, immune SPF chicken with expressing the IBDV recombinant VP 2 albumen that obtains.Gather chicken serum, indirect elisa method detects neutralizing antibody, and the result shows that the infectious bursal disease VP2 genetic engineering subunit vaccine that obtains among the present invention can effectively induce body to produce humoral immunoresponse(HI).In addition, challenge test also shows, IBDV recombinant VP 2 protein immunization SPF chicken can produce the immunoprotection that opposing virulent (vvIBDV) cause death to attack (immune group chicken survival rate more than 90%, not immune group chicken survival rate 15%).
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used for explanation the present invention and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example is write according to normal condition such as J. Pehanorm Brooker etc. usually, molecular cloning experiment guide, Science Press, the condition described in 2002, or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise per-cent and umber calculate by weight.
Unless otherwise defined, the same meaning that employed all specialties and scientific words and one skilled in the art are familiar with in the literary composition.In addition, any method similar or impartial to described content and material all can be applicable among the present invention.The usefulness that better implementation method described in the literary composition and material only present a demonstration.
Material and reagent
Infectious bursal disease highly virulent strain (vvIBDV) has separated self-infection the sick chicken of the superpower virus of infections chicken cloacal bursa (picking up from the Haining chicken house).
E. coli jm109 is available from Dalian Bao Bio-Engineering Company, F-strain Pichia.Pastoris X-33 (His-Mut+), expression vector pPICZ α A and microbiotic Zeocin
TMAvailable from Invitrogen company.
Restriction enzyme, the T4DNA ligase enzyme, high-fidelity Taq archaeal dna polymerase, the dna molecular quality standard is all available from Dalian Bao Bio-Engineering Company.
Preparation substratum yeast extract, lactoalbumin hydrolysate, YNB, vitamin H philosophy are available from Britain OXIOD company and Sigma company.Other reagent is import or domestic analytical pure product.
The acquisition of embodiment 1, goal gene
1, the extracting of the total RNA of viral genome
Fabricius bursa tissue is taken from the SPF chicken of infected chicken Very virulent infectious bursa disease virus (vvIBDV), fabricius bursa tissue is ground and homogenate freeze thawing three times.Get 0.02g and be organized in the 1.5mL EP pipe that DEPC processed, add 1mL Trizol, mixing, room temperature is placed 5min.Add 200 μ L chloroforms (generally adding in 0.2mL chloroform/mL Trizol ratio) in the sample of having processed, jog 15 seconds, ice bath 3min.4 ℃, the centrifugal 15min of 12,000rpm.Get the colourless water of supernatant in new EP pipe (DEPC pre-treatment), add the Virahol of 500 μ L precoolings, mixing, room temperature leaves standstill 10min.4 ℃, 12,000rpm, centrifugal 10min.Carefully abandon supernatant, add 1mL 95% (v/v) ethanol (newly preparing with DEPC water), slight mixing.4 ℃, the centrifugal 5min of 7500rpm.Abandon supernatant, blot liquid, vapour is done precipitation 5-10min.Add at last the DEPC pre-treatment without the distilled water 15-20 μ L of RNAase, beat and spare, the total RNA of dissolving in 55-60 ℃ of water-bath ,-20 ℃ or-80 ℃ save backup.The total RNA of genome of Here it is IBDV.
2, the clone of design of primers and goal gene
(1) design of primers
With reference to the VP2 gene order of having reported, use Oligo5.0 software at gene upstream and downstream design pair of primers.Introduce the cutting sequence of restriction enzyme site XhoI and Proteinase K EX2 in the upstream primer, introduce NotI restriction enzyme site and terminator codon TAA in the downstream primer.Primer sequence is respectively:
Upstream primer: 5 '-CA
CTCGAG 
ATGACAAACCTGCAA-3 ' (SEQ ID NO:2);
Downstream primer: 5 '-TA
GCGGCCGC 
CCTTAGGGCCCGGATTATGT-3 ' (SEQID NO:3).
Underscore is respectively XhoI, Not I restriction enzyme site; Italic is the cutting sequence of Proteinase K EX2; Be the reverse sequence of terminator codon (TAA) in the square frame.Above structure reason one: when recombinant protein by signal peptide guiding when emiocytosis is to born of the same parents, after signal peptide Protein cleavage enzyme (this Protein cleavage enzyme is natural to be present in the yeast body) cutting, can residual unnecessary amino acid at the N of target protein end, thus its natural immunity originality do not affected.Reason two: in downstream primer, add the TAA terminator codon, so that recombinant protein is only expressed VP2 albumen, and without c-myc epitope epitope and the hexahistidine tag polyhistidine tag of yeast vector self, make target protein keep natural radioactivity.Use the gene order that obtains behind the above primer amplification to see Fig. 1 (SEQ ID NO:1).
(2) goal gene clone
At first, take the total RNA of the viral genome of said extracted as template, utilize reverse transcription test kit Primer Script 1
StStrand cDNA Synthesis kit obtains cDNA, concise and to the point process is as follows: get the aseptic EP pipe that a DEPC processes, add one by one in order reaction solution, respectively Random 6Primers 1 μ L, dNTP Mixture (10mM each) 1 μ L, total RNA 8 μ L, behind above-mentioned reaction solution mixing, 65 ℃, 5min (carrying out in the PCR instrument), then chilling on ice adds following reagent: 5xRT damping fluid 4 μ L again in above-mentioned mixed solution, RNase Inhibitor (40U/ μ L) 0.5 μ L, Primer Script
TMRTase 1 μ L, ddH
2Behind the O 4.5 μ L mixings, in the PCR instrument, react by following program: 30 ℃, 10min; 42 ℃, 60min; 72 ℃, 15min; 4 ℃ of placements.
Then, the cDNA that obtains take above-mentioned reverse transcription again carries out pcr amplification as template.Reaction system is: ddH
2O 11.8 μ L, 10 * Taq PCR damping fluid, 2 μ L, d NTP 2 μ L, upstream primer 1 μ L, downstream primer 1 μ L, RT product 2 μ L, Taq DNA polymerase 10 .2 μ L.The PCR response procedures is: behind 95 ℃ of thermally denature 5min, and by 94 ℃ of sex change 50s, 56 ℃ of annealing 60s, 72 ℃ are extended 90s, carry out 30 circulations, in 72 ℃ of insulation 10min, at last 4 ℃ of placements.The purpose fragment that pcr amplification obtains reclaims through the rubber tapping of 1% agarose gel electrophoresis, carries out purifying again.
The structure of embodiment 2, recombinant expression plasmid and evaluation
Goal gene VP2 behind the above-mentioned purifying and expression vector pPICZ α A are carried out double digestion with XhoI/NotI respectively, reclaim purifying through 1% sepharose, carry out ligation with the T4 ligase enzyme, the recombinant expression plasmid called after pPICZ α A-VP2 that obtains.This recombinant expression plasmid is transformed escherichia coli jm109 competent cell again.In containing the less salt LB substratum of microbiotic Zeocin, separate mono-clonal.Test kit extracts plasmid, utilize XhoI/NotI that pPICZ α A-VP2 recombinant plasmid is carried out double digestion and identify (size is respectively the gene fragment of 3.6kb and 1.4kb) (seeing Fig. 2), utilize 5 of pPICZ α A carrier two ends ', 3 ' AOX1 primer (amplification obtains approximately 2.0kb gene fragment) and goal gene upstream and downstream primer (amplification obtains approximately 1.4kb gene fragment) carry out PCR and identify (seeing Fig. 3,4).Deliver to the order-checking of Invitrogen company with identifying correct recombinant plasmid.
Sequencing result shows, the goal gene sequence is entirely true, meets desired design, and it is also errorless to be inserted into the direction of carrier, coincide with the carrier reading frame.
Embodiment 3, recombinant plasmid electricity turn the screening of pichia spp and high resistance restructuring yeast strains
(1) linearizing of recombinant plasmid and purifying thereof
Recombinant plasmid pPICZ alpha A-VP2 SacI single endonuclease digestion, 37 ℃ of water-baths 2 hours, then 65 ℃ 20 minutes, make enzyme deactivation.Add isopyknic phenol/chloroform mixing, centrifugal two minutes of 12000rpm, supernatant liquor adds the dehydrated alcohol of 2.5 times of volumes and the 3M sodium acetate of 1/10 volume, and mixing is placed 10 minutes with precipitation DNA for-20 ℃.Centrifugal 5 minutes of 4 ℃, 12000rpm, supernatant discarded, precipitation is with 80% (v/v) washing with alcohol, the ddH that sterilizes with 10 μ L after the drying at room temperature
2O is resuspended, is the linearizing recombinant plasmid of purifying, deposit-20 ℃ for subsequent use.
(2) preparation of pichia spp competent cell
Competence preparation method is as follows: inoculation pichia pastoris X-33 mono-clonal is in 3mL YPD liquid nutrient medium, and 30 ℃, 270rpm shaking table shaking culture spend the night.In this bacterium 1: 100 inoculation 100mLYPD liquid nutrient medium that spends the night, 30 ℃, 270rpm shaking table shaking culture are to OD
600=1.3-1.5 approximately needs 20 hours.Centrifugal 5 minutes of 4 ℃, 1500g.The ddH of 0 ℃ of 100mL of cell precipitation, sterilization
2O is resuspended.With centrifugal 5 minutes of above-mentioned step 4 ℃, 1500g, cell precipitation was with the ddH of 0 ℃ of 50mL, sterilization
2O is resuspended.Centrifugal 5 minutes of 4 ℃, 1500g, cell precipitation is resuspended with the 1M sorbitol (Sorbitol Powder) of 0 ℃ of 4mL, sterilization.With above-mentioned step centrifuged deposit be resuspended in again 0 ℃ of 0.2mL, the sterilization 1M sorbitol in, to final volume be 0.3mL, namely make yeast X-33 competent cell.Place it on ice and used the same day.
(3) electricity changes pichia spp over to
Through condition optimizing, this experiment takes following electricity to turn parameter: 0.2cm electricity revolving cup, and voltage 2.0KV, electric shock time 5ms, best plasmid concentration is that 7ug is to 10ug (volume is controlled at 10 μ L), competent cell 100 μ L.
Electricity turns experiment and carries out in the BIO-RAD electroporation, adds fast 1mL 1Msorbitol (Sorbitol Powder) after electric the conversion, recovers 1 hour for 30 ℃, coating contains the YPDS flat board of Zeocin again, flat board is inverted, is placed in the constant incubator, cultivated 2-3 days for 30 ℃.
(4) screening of high resistance (copy) recombinant bacterial strain
Contain Zeocin among the carrier pPICZ α A
TMResistant gene is according to bibliographical information restructuring yeast strains opposing Zeocin
TMAbility be directly proportional with its integrated plasmid copy number, therefore, with containing different Zeocin
TMThe agar plate of concentration (100 μ g/mL-3000 μ g/mL) can rapid screening contain the strain of multiple copied gene transformation.
Concrete operation method is: use the toothpick picking of sterilization at Zeocin
TMConcentration is that the electricity of 100 μ g/mL turns the single bacterium colony on the flat board, and it is distinguished dibbling at Zeocin
TMConcentration is respectively on the YPDS flat board of 1000 μ g/mL, 2000 μ g/mL, 3000 μ g/mL.Flat board is inverted, is placed in the constant incubator, cultivated 2-3 days for 30 ℃.Press bacterium colony growing way size, from big to small picking Zeocin
TMConcentration is 20 strain mono-clonals on the YPDS flat board of 3000 μ g/mL.With this 20 strain mono-clonal Zeocin that rules respectively
TMConcentration is that the YPDS of 100 μ g/mL is dull and stereotyped, with the purifying mono-clonal.
(5) PCR identifies positive colony and phenotype thereof
The mode of exogenous origin gene integrator yeast chromosomal has two kinds, be respectively displacement restructuring and the restructuring that intersects, two kinds of different bacterial strains of the corresponding generation of these two kinds of recombination forms are respectively that the slow type bacterial strain of methyl alcohol utilization (Muts) and methyl alcohol utilize Quick-type bacterial strain (Mut+).Identify this two kinds of phenotypes, comparatively easy identification method is PCR method.Utilize the two ends primer 5 ' AOX1:5 ' of expression vector-GACTGGTTCCAATTGACAAGC-3 ' (SEQ ID NO:4), 3 ' AOX1:5 '-GCAAATGGCATTCTGACATCC-3 ' (SEQ ID NO:5), carry out PCR and identify.If pcr amplification product is single fragment, size is 2.0kb, then is slow growth type transformant, slowly utilizes methyl alcohol.If pcr amplification product except the 2.0kb fragment, also has the gene fragment of a treaty 2.2kb size, then is the Quick-type transformant, can utilize fast methyl alcohol (seeing Fig. 5).Another advantage of this PCR authentication method is, its can detect positive colony simultaneously, has simplified testing sequence.Select the Quick-type transformant.
The used template of above-mentioned PCR reaction, generally commonly used is the direct extraction pastoris genomic dna, but it is comparatively consuming time loaded down with trivial details to obtain by this method pcr template, and not only uneconomical for a large amount of transformant screening, and also workload is very large.The present invention has studied more easy method, directly adopts yeast colony to carry out PCR.Yeast colony is made its cell walls cracking through cold heat-treating methods, change some moietys and the volume of common PCR reaction solution again, practice shows, this method accuracy rate is high, also saves time very much effectively.
The abduction delivering of embodiment 4, recombination yeast engineering bacteria and the evaluation of target protein
(1) shaking flask abduction delivering
Picking is cloned in (250mL shaking flask) among the 25mL BMGY, 30 ℃, 270rpm, cultivate approximately 16-18 hour to OD600 be 2-6,2000g, 5min, the centrifugal collecting cell precipitation is resuspended in (500mL shaking flask) among the 100mLBMMY with precipitation, the beginning abduction delivering.Adding final concentration every 12-24 hour is the methyl alcohol of 0.5% (v/v).Be cultured to 120h and collect fermented liquid, the centrifugal 2-3 of maximum speed of revolution minute, abandon precipitation, supernatant is kept at first the very low temperature refrigerator-freezer.
In shaking flask abduction delivering process of the test, the inventor is optimized each expression condition, carries out abduction delivering with the condition of optimum.The expression condition of optimizing is: the pH value of the growth medium BMGY of engineering bacteria is 6.0, and vegetative period, temperature was 30 ℃; The pH value of inducing culture BMMY is 6.5, and inducing temperature is 28.5 ℃, and inductor methyl alcohol final concentration is 0.5%, and expression amount is up to 0.766mg/ml.By consulting literatures learns, this expression amount is the high expression level amount of utilizing at present that Yeast system carries out that recombinant vaccine institute obtains.
(2) evaluation of expressing protein
Ordinary method carries out SDS-PAGE and the Western-blot qualification result shows, the recombinant plasmid expression that in Yeast system, succeeds, and target protein and IBDV polyvalent antibody occur can specific reaction, confirmed that target protein has immunoreactivity.The results are shown in Figure 6-7.
The preparation of embodiment 5, VP2 protein subunit vaccine and animal immune experiment
Be that 2: 3 (volume ratio) carries out emulsification according to the water-oil ratio example, namely two parts of recombinant protein fermentation supernatants (contain 0.5% (v/v) tween-80, protein concentration is 0.766mg/ml) originally (white oil is fought available from Shandong and is moistened Industrial Technology Co., Ltd to add 3 parts of white oil departments, department this available from Beijing Xi Kai Innovation Co., Ltd) fully emulsified evenly, the VP2 protein subunit vaccine.
Get 40 of 7-9 age in days SPF chickens, divide three groups, 10 every group, every immunizing dose is 0.5mL, and immunization ways is subcutaneous injection.The A group is VP2 protein subunit vaccine of the present invention, and the B group is conventional infections chicken cloacal bursa freeze dried vaccine (fabricius bursa B87 freeze-dried vaccine), and C organizes negative control.Carry out two after 15 days and exempt from (dosage is every chicken of 0.5mL with for the first time immunity).Week about blood sampling between duration of immunity, indirect elisa method is measured the neutralizing antibody level.The antibody titers measurement result of A group is seen Fig. 8.
The result shows, the VP2 protein subunit vaccine can stimulate body to produce the protectiveness neutralizing antibody of high titre fully among the present invention.
Embodiment 6, challenge test
With VP2 protein subunit vaccine immunity SPF chicken of the present invention, rear 10 days IBDV highly virulent strains of immunity are attacked poison for the second time such as the method for embodiment 5, attack poison and observe protection ratio and the variation of fabricius bursa tissue in rear 10 days.
Attack rear 10 days test chicken fabricius bursa histological examination results of poison: the equal not damaged of the lymph follicle of the fabricius bursa, cortex and medullary substance boundary are clear, and fabricius bursa one-piece construction is normal.
Attacked poison rear 10 days, immune chicken group protection ratio is 95%, and not immune chicken group protection ratio only is 15%.This has shown, the IBDV recombinant VP 2 protein subunit vaccine that the present invention expresses can be protected the lethal hit of immune chicken opposing highly virulent strain fully.
All quote in this application as a reference at all documents that the present invention mentions, just as each piece document is quoted separately as a reference.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (5)
1. the method for a recombinant expressed Infectious bursal disease virus VP2 subunit is characterized in that, the method comprises:
(1) provides the VP2 protein subunit encoding gene of transformation; Wherein, the cutting sequence of adding Proteinase K EX2 before 5 ' end initiator codon ATG of described VP2 protein subunit encoding gene is 3 ' end interpolation termination codon subsequence of described VP2 protein subunit encoding gene;
(2) (1) described VP2 protein subunit encoding gene is inserted into the α of Yeast expression carrier pPICZ α A-factor signal coding sequence downstream, obtains recombinant expression vector;
(3) the recombinant expression vector electricity with (2) transforms the pichia pastoris X-33 bacterial strain, obtains the Pichia pastoris of restructuring; The electricity conversion condition is: 0.2cm electricity revolving cup, and voltage 2.0KV, electric shock time 5ms, plasmid concentration is 7-10 μ g/10 μ L, competent cell 100 μ L; Add fast the 1mL1M Sorbitol Powder after electric the conversion, recovered 1 hour for 30 ℃;
(4) Pichia pastoris of the restructuring of (3) is carried out recombinant expressed, expression condition is: adopt inducing culture BMMY, the substratum pH value is 6.5 ± 0.5, and inducing temperature is 28.5 ± 1 ℃, and the methyl alcohol final concentration is 0.5 ± 0.2% (v/v); From express supernatant, obtain Infectious bursal disease virus VP2 subunit;
In the step (1), also before 5 ' end of the cutting sequence of described Proteinase K EX2, add XhoI restriction enzyme site sequence; 3 ' end described termination codon subsequence adds NotI restriction enzyme site sequence;
The sequence of the VP2 protein subunit encoding gene of described transformation is shown in SEQ ID NO:1.
2. the method for claim 1 is characterized in that, in the step (2), described VP2 protein subunit encoding gene is inserted between the XhoI/NotI restriction enzyme site of Yeast expression carrier pPICZ α A.
3. a method for preparing the infectivity bursa of Fabricius virus VP 2 protein subunit vaccine is characterized in that, the method comprises:
Obtain Infectious bursal disease virus VP2 subunit with method claimed in claim 1, carry out emulsification, obtain the infectivity bursa of Fabricius virus VP 2 protein subunit vaccine.
4. method as claimed in claim 3 is characterized in that, described emulsification be with described Infectious bursal disease virus VP2 subunit originally mix with white oil department, emulsification.
5. polynucleotide that are used for recombinant expressed Infectious bursal disease virus VP2 subunit is characterized in that, its nucleotide sequence is shown in SEQ ID NO:1.
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| CN104353070B (en) * | 2014-11-05 | 2017-04-19 | 中山大学 | Genetic engineering subunit vaccine of chicken infectious bronchitis virus and preparation method 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 |
| CN111662364A (en) * | 2020-06-28 | 2020-09-15 | 山西农业大学 | Infectious bursal disease virus VP2 protein and coding gene and application thereof |
| CN112375125B (en) * | 2020-11-23 | 2021-09-28 | 东北农业大学 | Polypeptide and application thereof in preventing infectious bursal disease of chicken |
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