CN102277374A - 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 mass mortality, cause immunosuppression simultaneously, make chicken group vaccine inoculation immuning failure and secondary, concurrent disease roll up, cause serious economy loss.This disease has become serious, the most thorny transmissible disease of harm China's poultry husbandry, does not have special effect medicine therapeutic at present.
Means of prevention commonly used at present is vaccine inoculation, 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, make traditional vaccine can not control that it is popular, bring 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 in mass-producing poulty house; the demand of annual bursal disease vaccine also increases rapidly; therefore be badly in need of developing a kind of product safely and effectively, 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 reorganization development reaches its maturity, and recombinant vaccine has been the main direction that develops both at home and abroad.Comprehensive relatively range gene engineered vaccine wherein has more special advantages with protein subunit vaccine especially.Protein subunit vaccine never contains malicious composition alive, and production and use do not have the poison of loosing possibility, have eliminated contamination of heavy, is the worth safe vaccine of actively promoting the use of.
Formerly patent " 200410080065.8, a kind of preparation method of genetic engineering subunit vaccine of infectious bursal disease and application " in, subunit vaccine can stimulate body to produce the attack of neutralizing antibody and effectively opposing virus, precipitation is carried out numerous and diverse programs such as repeatedly freeze thawing ultrasonication but prokaryotic expression system needs pair cell, 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, the synthetic back of complete genome sequence in yeast, expressed.But the residual one section sequence of expressing in this patent that has on the carrier of target protein C end does not have natural sex, and has influenced its immunogenicity.
Therefore, still need to improve the technology of preparing of gene engineering subunit vaccine of chicken's infectious bursal disease,, obtain the strong vaccine of immunogenicity to improve expression amount.
Summary of the invention
The object of the present invention is to provide the preparation and the application of a kind of infectious bursal disease gene engineered subunit (VP2) vaccine.
In a first aspect of the present invention, a kind of method of recombinant expressed chicken infectivity bursa of Fabricius virus VP 2 protein subunit is provided, this 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 α-factor signal coding sequence downstream of Yeast expression carrier pPICZ α A, obtains recombinant expression vector;
(3) recombinant expression vector with (2) transforms pichia spp, obtains the pichia spp cell of reorganization;
(4) the pichia spp cell of the reorganization of (3) is carried out recombinant expressed, from express supernatant, obtain the chicken infectivity bursa of Fabricius virus VP 2 protein 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; The electric back that transforms adds 1mL 1M Sorbitol Powder fast, recovers 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, this method comprises:
Obtain the chicken infectivity bursa of Fabricius virus VP 2 protein subunit with described method, carry out the emulsive step, obtain the infectivity bursa of Fabricius virus VP 2 protein subunit vaccine.
In another preference, with described chicken infectivity bursa of Fabricius virus VP 2 protein subunit originally mix with white oil department, emulsification.
In another aspect of this invention, provide a kind of polynucleotide that are used for recombinant expressed chicken infectivity bursa of Fabricius virus VP 2 protein subunit, its nucleotide sequence is shown in SEQ ID NO:1.
In another aspect of this invention, provide a kind of primer that is used to obtain described polynucleotide, the nucleotide sequence of described primer is shown in SEQ ID NO:2 and SEQ ID NO:3.
Others of the present invention are because the disclosure of this paper is conspicuous 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, recombinant expression plasmid double digestion identify that 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 proteic recombinant expression method of chicken infectivity bursa of Fabricius virus VP 2 through deep research, has effectively improved the proteic expression output of VP2, has guaranteed the consistence of expression product and native protein.Method technology of the present invention is simple, with low cost, can obtain the chicken infectivity bursa of Fabricius virus VP 2 protein subunit vaccine that can use safely efficiently and stably.
Expression system
In the past, people adopted prokaryotic expression system to express VP2 albumen morely, expression process complexity, and difficult operation, and protein yield is difficult to improve.Therefore, the inventor is devoted to develop new VP2 protein expression system, in the hope of raising output, and obtains the good albumen of immunogenicity, 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 zymic advantage is very obvious.The pichia methanolica expression system can be translated post-treatment accurately with the recombinant protein of expressing and modify, and approaches 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 technology 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 bacterium.
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 the proteic expression constructs of VP2.
α-factor signal peptide on the Yeast expression carrier pPICZ α A comprises one section spacer peptide of being made up 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 proteic correct cutting and secrete most important, but sometimes because the cutting vigor difference of above-mentioned two kinds of gene products, can produce different cleavage sites at the N of foreign protein end, under 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, promptly produced so-called signal peptide of yeast and cut incomplete phenomenon, this may influence 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, preceding two restriction enzyme sites that aminoacid sequence is restriction enzyme XhoI, latter two aminoacid sequence is the cutting sequence of embrane-associated protein enzyme KEX2, promptly, VP2 albumen has only merged the cutting sequence A AA-AGA (Lys-Arg) of one of them Proteinase K EX2 among the present invention, She Ji purpose 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 influence its antigenicity, keep proteic integrity and natural radioactivity; Its two, add terminator TAA at VP2 gene 3 ' end, thereby removed two sections sequence c-myc epitope and polyhistidine tag on the carrier, make that the target protein of expressing can be not residual except other the unnecessary sequences self equally.
Make a general survey of prior art, not appearing in the newspapers 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, direct relation is arranged with above-mentioned gene order transformation.
Expression method
The method of recombinant expressed chicken infectivity bursa of Fabricius virus VP 2 protein subunit of the present invention mainly comprises:
(1) provides the VP2 protein subunit encoding gene of transformation;
(2) (1) described VP2 protein subunit encoding gene is inserted into α-factor signal coding sequence downstream of Yeast expression carrier pPICZ α A, obtains recombinant expression vector;
(3) recombinant expression vector with (2) transforms pichia spp, obtains the pichia spp cell of reorganization;
(4) the pichia spp cell of the reorganization of (3) is carried out recombinant expressed, from express supernatant, obtain the chicken infectivity bursa of Fabricius virus VP 2 protein 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 changeing because of electricity, the concentration of electric commentaries on classics time and plasmid, 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 repeatedly testing, the inventor has obtained preferable electricity changes parameter: plasmid concentration is 7-10ug/10 μ L, competent cell 100 μ L; The electric back that transforms adds lmL 1M Sorbitol Powder fast, recovers 1 hour for 30 ℃.In when operation, the last amount that adds 1M sorbitol of control, make 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 change effect can be better.
Optimization with the commentaries on classics condition that powers 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 protein expression at normal temperatures, 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 spp 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.Shake a bottle abduction delivering test and obtain 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) are identified and are shown that recombinant VP 2 albumen has the biological activity and the immunogenicity of chicken infectivity bursa of Fabricius virus native protein.
Vaccine production
Adopt the VP2 albumen of the recombinant expressed acquisition of aforesaid method of the present invention, can be used to prepare vaccine through behind the 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 to the present invention is described 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. Sa nurse 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 preferable 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 etc. are respectively available from Britain OXIOD company and Sigma company.Other reagent is import or homemade 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 infectious bursal disease highly virulent strain (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 handled, add 1mL Trizol, mixing, room temperature is placed 5min.Add 200 μ L chloroforms (generally adding) in the sample of having handled, jog 15 seconds, ice bath 3min in 0.2mL chloroform/mL Trizol ratio.4 ℃, 12, the centrifugal 15min of 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), slight mixing with DEPC water.4 ℃, the centrifugal 5min of 7500rpm.Abandon supernatant, blot liquid, vapour is done precipitation 5-10min.Adding the DEPC pre-treatment does not at last have the distilled water 15-20 μ L of RNAase, beat even, the total RNA of dissolving in 55-60 ℃ of water-bath ,-20 ℃ or-80 ℃ of preservations are standby.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 and design a pair of primer in the gene upstream and downstream.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; In the square frame is the reverse sequence of terminator codon (TAA).More than make up reason one: when recombinant protein by the signal peptide guiding when emiocytosis is to born of the same parents, after signal peptide albumen nickase (this albumen nickase 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 influenced.Reason two: in downstream primer, add the TAA terminator codon, make recombinant protein only express VP2 albumen, and do not have the 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, total RNA is a template with the viral genome of said extracted, utilizes 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 handles, add reaction solution in order one by one, be 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), chilling on ice adds following reagent: 5xRT damping fluid 4 μ L again in above-mentioned mixed solution then, 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 with above-mentioned reverse transcription is a template again, carries out pcr amplification.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, carry out ligation with the T4 ligase enzyme, the recombinant expression plasmid called after pPICZ α A-VP2 that obtains through 1% sepharose.With this recombinant expression plasmid transformed into 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) (see figure 2), utilize 5 of pPICZ α A carrier two ends ', 3 ' AOX1 primer (amplification obtains about 2.0kb gene fragment) and goal gene upstream and downstream primer (amplification obtains about 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 that target gene sequences 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 change 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 deposit D NA 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 ℃ standby.
(2) preparation of pichia spp competent cell
Competence preparation method is as follows: inoculation pichia spp 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, promptly 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 change parameter: 0.2cm electricity revolving cup, voltage 2.0KV, and 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 changes experiment and carries out in the BIO-RAD electroporation, and the electric back that transforms adds 1mL 1Msorbitol (Sorbitol Powder) fast, recovers 1 hour for 30 ℃, and coating contains the YPDS flat board of Zeocin again, and flat board is inverted, and is placed in the constant incubator, cultivates 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 multiple copied foreign gene transformant.
Concrete operation method is: the toothpick picking of using sterilization is at Zeocin
TMConcentration is that the electricity of 100 μ g/mL changes 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 the YPDS flat board of 100 μ g/mL, 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, is respectively the displacement reorganization and the reorganization that intersects, and two kinds of different bacterial strains of the corresponding generation of these two kinds of recombination forms are respectively slow type bacterial strain of methyl alcohol utilization (Muts) and the quick type bacterial strain of methyl alcohol utilization (Mut+).Identify this two kinds of phenotypes, comparatively easy identification method is a PCR method.Utilize two ends primer 5 ' AOX1:5 '-GACTGGTTCCAATTGACAAGC-3 ' (SEQ ID NO:4), the 3 ' AOX1:5 '-GCAAATGGCATTCTGACATCC-3 ' (SEQ ID NO:5) of expression vector, 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 quick type transformant, can utilize the methyl alcohol (see figure 5) fast.Another advantage of this PCR authentication method is that it can detect positive colony simultaneously, has simplified testing sequence.Select quick type transformant.
The used template of above-mentioned PCR reaction, general is direct extracting pastoris genomic dna than what use always, but it is comparatively consuming time loaded down with trivial details to obtain pcr template by this method, and not only uneconomical for a large amount of transformant screening, and also workload is very big.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 height also saves time effectively very much.
The abduction delivering of embodiment 4, recombination yeast engineering bacteria and the evaluation of target protein
(1) shakes a bottle abduction delivering
Picking is cloned in 25mL BMGY (250mL shakes bottle), 30 ℃, 270rpm, cultivate about 16-18 hour to OD600 be 2-6,2000g, 5min, the centrifugal collecting cell precipitation is resuspended in precipitation among the 100mLBMMY (500mL shake bottle), begins 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 the very low temperature refrigerator-freezer earlier.
Shaking a bottle abduction delivering process of the test, the inventor optimizes 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.Consult document and learn, this expression amount is 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 of in Yeast system, succeeing, and target protein and IBDV polyvalent antibody take place 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
According to the water-oil ratio example is that 2: 3 (volume ratio) carries out emulsification, promptly 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 immunity for the first time).Blood sampling week about 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 that 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, back 10 days IBDV highly virulent strains of immunity are attacked poison for the second time as the method for embodiment 5, attack poison and observe protection ratio and the variation of fabricius bursa tissue in back 10 days.
Attack back 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 back 10 days, immune chicken group protection ratio is 95%, and not immune chicken group protection ratio only is 15%.This has shown that 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 quoted as a reference separately as each piece document.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 institute restricted portion equally.
Claims (10)
1. the method for a recombinant expressed chicken infectivity bursa of Fabricius virus VP 2 protein subunit is characterized in that, this 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 α-factor signal coding sequence downstream of Yeast expression carrier pPICZ α A, obtains recombinant expression vector;
(3) recombinant expression vector with (2) transforms pichia spp, obtains the pichia spp cell of reorganization;
(4) the pichia spp cell of the reorganization of (3) is carried out recombinant expressed, from express supernatant, obtain the chicken infectivity bursa of Fabricius virus VP 2 protein subunit.
2. the method for claim 1 is characterized in that, in the step (1), also adds XhoI restriction enzyme site sequence before 5 ' end of the cutting sequence of described Proteinase K EX2; 3 ' end described termination codon subsequence adds NotI restriction enzyme site sequence.
3. method as claimed in claim 2 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.
4. method as claimed in claim 2 is characterized in that, the sequence of the VP2 protein subunit encoding gene of described transformation is shown in SEQ ID NO:1.
5. the method for claim 1 is characterized in that, described pichia spp is the X-33 bacterial strain.
6. the method for claim 1 is characterized in that, the recombinant expression vector electricity is transformed pichia spp, and electric conversion condition is: 0.2cm electricity revolving cup, and voltage 2.0KV, electric shock time 5ms, plasmid concentration is 7-10ug/10 μ L, competent cell 100 μ L; The electric back that transforms adds 1mL 1M Sorbitol Powder fast, recovers 1 hour for 30 ℃.
7. the method for claim 1 is characterized in that, 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).
8. a method for preparing the infectivity bursa of Fabricius virus VP 2 protein subunit vaccine is characterized in that, this method comprises:
Obtain the chicken infectivity bursa of Fabricius virus VP 2 protein subunit with the described method of claim 1, carry out the emulsive step, obtain the infectivity bursa of Fabricius virus VP 2 protein subunit vaccine.
9. method as claimed in claim 8 is characterized in that, with described chicken infectivity bursa of Fabricius virus VP 2 protein subunit originally mix with white oil department, emulsification.
10. polynucleotide that are used for recombinant expressed chicken infectivity bursa of Fabricius virus VP 2 protein subunit is characterized in that, its nucleotide sequence is shown in SEQ ID NO:1.
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| 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 |
| CN112375125A (en) * | 2020-11-23 | 2021-02-19 | 东北农业大学 | Novel polypeptide and application thereof in preventing infectious bursal disease of chicken |
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