CN103585625B - A kind of porcine epizootic diarrhea recombinant baculovirus genetic engineering subunit vaccine and preparation method thereof and application - Google Patents
A kind of porcine epizootic diarrhea recombinant baculovirus genetic engineering subunit vaccine and preparation method thereof and application Download PDFInfo
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Abstract
The invention belongs to biovaccine preparing technical field, be specifically related to a kind of porcine epizootic diarrhea recombinant baculovirus genetic engineering subunit vaccine and preparation method thereof and application.The present invention chooses the S1 gene of the new epidemic isolates of current PE DV and M gene is reference sequences, baculovirus expression system is adopted to express S1 albumen or part S1 albumen and M albumen, the recombiant protein of acquisition is prepared into subunit vaccine, for effectively controlling the generation of porcine epizootic diarrhea.The porcine epizootic diarrhea genetic engineering subunit vaccine that the method for the invention obtains solves the defect that current Porcine epidemic diarrhea virus traditional vaccine exists, can be used for the relevant disease that prevention and therapy Porcine epidemic diarrhea virus infects and causes, be applicable to too prepare the envelope antigen detecting Porcine epidemic diarrhea virus antibody ELISA test kit.
Description
Technical field
The invention belongs to biovaccine preparing technical field, be specifically related to a kind of porcine epizootic diarrhea recombinant baculovirus genetic engineering subunit vaccine and preparation method thereof and application.
Background technology
Porcine epizootic diarrhea (Porcineepidemicdiarrhea, PED) be a kind of acute high degree in contact infectious intestinal disease, caused by Porcine epidemic diarrhea virus (Porcineepidemicdiarrheavirus, PEDV), its Clinical symptoms is watery diarrhea, vomiting and dehydration.The equal susceptible of pig of each age in days, especially suckling pig.PED worldwide extensively distributes, and the states such as Britain, Belgium, France, Hungary, Canada report the generation of primary disease in succession.It is reported, PED occurred quite serious in recent years in Asian countries, being very popular all appearred in Japan, Korea S, Thailand, and mortality rate is high, and harm is serious, causes huge economic loss to pig industry.
China was separated to PEDV first in 1980, and later many areas have reported that primary disease occurs in succession, and it is little mainly to distribute at winter-spring season the harm caused.From 2010 so far, in China south China, part province, North China broken out serious porcine epizootic diarrhea, the piglet death (Sunetal, 2012) more than 1,000,000.Show as piglet watery diarrhea, vomiting, high mortality in 7 ages in days, selected swine farms repeated infection such as all to be fallen ill throughout the year at the new epidemic characteristic.In addition, up-to-date research shows also to carry out vertical transmission by milk.At present traditional inactivated vaccine, Attenuate vaccine immunity inoculation are mainly taked for the prevention and control of this disease, but the immune protective efficiency produced all does not reach desirable effect.Cause the main cause of immuning failure to have: (1) virus titer is low, cause antigenic content not enough.PEDV is difficult to separation and Culture, impel it to breed in Vero cell to go down to posterity, but virus titer can only maintain 1 × 10 even if add pancreatin in nutritional solution
3.01tCID
50about/ml.(2) strain variation.PEDV spike protein (S protein) is the immune protein that induction body produces protectiveness neutralizing antibody, by S gene code.Now report, current China is newly separated PEDV strain S gene and there occurs variation, Phylogenetic tree analysis point out new isolated strain and Thailand's isolated strain relation nearer, (Liu Xiaozhen etc. comparatively far away from vaccine strain CV777 genetic affinity, 2012), the current conventional P ED vaccine of China is all adopt this vaccine strain.Because existing vaccine effect in prevention and control PED is undesirable, the prevention and control problem of PED is made to seem very thorny.Therefore, a kind of new generation vaccine that is safe, effective, reasonable price is developed extremely urgent to the Occurrence & epidemic controlling this disease.
PEDV belongs to coronaviridae (Coronaviridae) coronavirus genus (Coronavirus).The main structural protein of PEDV comprise: nucleocapsid protein (N protein) is that PEDV produces maximum virus proteins in infection cell, and structure is very conservative, therefore utilizes N protein to have good application prospect to set up PEDV diagnostic technique in molecular biology; Memebrane protein (M albumen) is the envelope protein that in PEDV, content is maximum, be made up of 226 aminoacid, have the biological action of three aspects: participate in the assembling of virion and sprout, stimulate body to produce alpha-interferon (α-IFN), antibody of its generation have when complement exists in and the ability of virus; Spike protein (S protein) is by the Spike Glycoprotein being positioned at virion surface of S gene code, is merged in the process invading host cell and mediate neutralizing antibody generation in infection host body all play important biomolecule effect after virion is combined with cell surface receptor by film.The polypeptide that S protein is made up of 1384 aminoacid, containing the individual potential glycosylation site of 27-29, is divided into 2 functional areas and S1(1-789 amino acids) and S2(790-1383 amino acids).Now study and confirmed to there is multiple linear epitope in S1 district, comprised neutralizing epitope district (ChangSHetal., 2002; Sun Dongbo etc., 2007).So M albumen and S protein are the important target protein in the aspect researchs such as PEDV recombinant vaccine, Virus entry mechanism and diagnostic techniques.
At present, researcher, in order to play the characteristic of S1 albumen and M albumen energy inducing antibodies, adopts prokaryotic expression system such as escherichia coli, lactobacillus, Salmonella to be expression vector, expresses antigen epitope genes (Liu Deng etc., 2010 of S1 district part; Dong Lina etc., 2008; Xu Lili etc., 2011).Also there is researcher to adopt escherichia coli to be vector expression M albumen (Gao Shenyang etc., 2007) simultaneously.But the equal Shortcomings part of above-mentioned expression strategy: S protein is glycosylated protein, prokaryotic expression system can not carry out corresponding modification and cause the immunogenicity of restructuring S1 albumen or the M albumen given expression to poor; Expression is generally not high causes on the low side being not enough to of Effective Antigens amount content to be prepared into subunit vaccine; Be all adopt single expression, do not adopt double expression(DE) system to play the biologic activity of S1 albumen and M albumen simultaneously.
Summary of the invention
The technical problem to be solved in the present invention is to overcome prior art selected part S1 gene or complete M gene, adopts prokaryotic expression system to carry out expressing the defect existed, provides a kind of porcine epizootic diarrhea recombinant baculovirus genetic engineering subunit vaccine.
Another object of the present invention is to provide a kind of preparation method of porcine epizootic diarrhea recombinant baculovirus genetic engineering subunit vaccine.Described method chooses the S1 gene of the new epidemic isolates of current PE DV and M gene is reference sequences, adopting baculovirus expression system to express S1 albumen and M albumen, the recombiant protein of acquisition being prepared into subunit vaccine, for effectively controlling the generation of porcine epizootic diarrhea.
Object of the present invention is achieved by the following technical programs:
A preparation method for porcine epizootic diarrhea recombinant baculovirus genetic engineering subunit vaccine, comprises the steps:
S1. the encoding gene of complete S1 albumen of Porcine epidemic diarrhea virus or the encoding gene S1p of the part S1 albumen of Porcine epidemic diarrhea virus and the encoding gene of entire M protein are cloned in baculovirus expression system and obtain recombinant baculovirus Bv-S1 or recombinant baculovirus Bv-S1p-M;
S2. recombinant baculovirus Bv-S1p-M or recombinant baculovirus Bv-S1 is carried out amplification culture, purification reclaims and obtains S1 albumen or S1p albumen and M albumen;
S3. can obtain after S1 albumen or S1p albumen and M albumen and pharmaceutically acceptable adjuvant fully being mixed and organize Baculovirus Gene engineering subunit vaccine;
Wherein the nucleotide sequence of the encoding gene S1p of the part S1 albumen of Porcine epidemic diarrhea virus is as shown in SEQIDNO:1; The nucleotide sequence of S1 full genome is as shown in SEQIDNO:2; The nucleotide sequence of M gene is as shown in SEQIDNO:3.
Particularly, described in S1, the construction method of recombinant baculovirus Bv-S1 is: be cloned in baculovirus transfer vector pFastbac-HTB by the encoding gene of the complete S1 albumen of Porcine epidemic diarrhea virus, obtain recombinant vector pFastbac-HTB-S1; Recombinant vector pFastbac-HTB-S1 is transformed into pre-containing in the DH10Bac competent cell of baculovirus skeleton plasmid, and by extracting recombinant vector after homologous recombination, recombinant vector again transfection Sf9 cell produces recombinant baculovirus Bv-S1.
Particularly, described in S1, the construction method of recombinant baculovirus Bv-S1p-M is: the encoding gene S1p of the part S1 albumen of Porcine epidemic diarrhea virus and the encoding gene of entire M protein are cloned in baculovirus transfer vector pFastbac-Dual, obtain recombinant vector pFastbac-Dual-S1p-M; Recombinant vector pFastbac-Dual-S1p-M is transformed into pre-containing in the DH10Bac competent cell of baculovirus skeleton plasmid, and by extracting recombinant vector after homologous recombination, recombinant vector again transfection Sf9 cell produces recombinant baculovirus Bv-S1p-M.
Amplification culture described in S2 is: be 1 ~ 2 × 10 by density
6after the recombinant baculovirus of cell/mL connects malicious infected insect cell, insect cell containing 0.1 ~ 0.5% FBS or BSA serum-free medium in, cultivate 72h for 28 DEG C.
Described in S2, purification is recovered as: the insect cell after centrifugal recovery S2 amplification culture, utilizes affinity chromatography medium Ni-NTA to carry out purification and reclaim destination protein.
Preferably, adjuvant described in S3 is oil emulsion adjuvant or aluminium glue adjuvant
The porcine epizootic diarrhea recombinant baculovirus genetic engineering subunit vaccine that method described above prepares.
The application of porcine epizootic diarrhea recombinant baculovirus genetic engineering subunit vaccine as above in prevention and control porcine epizootic diarrhea.
The invention has the beneficial effects as follows:
(1) the present invention chooses complete S1 protein gene and M protein gene, contains complete linear epitope (neutralizing epitope), and therefore induction produces strong for the ability of PEDV specific antibody.Obtain S1 district gene be up-to-date epidemic isolates gene, with China up-to-date announcement strain CH/FJND-3/2011(serial number: JN381492.1) nucleotide sequence homology be 98.76%.Therefore the expressed S1 albumen generation specific antibody produced has more specific aim and practicality.And the ability adopting the dual-expression vector in baculovirus expression system to possess S1 gene and M gene to express, adds expression of recombinant proteins amount thus significantly improves the Effective Antigens content in subunit vaccine simultaneously.
(2) the present invention adopts baculovirus to be expression vector, and be exogenous gene carrier because rod string design is one with baculovirus, insecticide and insect cell are the expression system of receptor.Compared with other expression systems, it has can hold larger exogenous gene insertion; Multiple gene can be expressed simultaneously; Expression is high; Expression product can carry out comprising glycosylation, phosphorylation, acyl group, signal peptide excision and peptide section the post translational processing of cutting Sum decomposition etc. modify; To vertebrates without advantages such as infectivities.Have good immunogenicity and higher expression by each antigen protein of the PEDV of baculovirus expression, expressed antigen protein can stimulate pig body to produce effective immunity, prevents the generation of PED better.
figure of description
Fig. 1. transfer vector pFastbac-Dual-S1p-M double digestion qualification result electrophoretogram; M:250bpMarker; 1.pFastbac-Dual empty control plasmid; 2. empty plasmid KpnI, Hind III; 3. transfer vector recombiant plasmid pFastbac-Dual-S1p-M; 4.pFastbac-Dual-S1p-MKpn I, Hind III double digestion product; 5.pFastbac-Dual-S1p-MXho I, Xba I double digestion; 6.2000bpMaker.
Fig. 2. recombinant vector Bacmid-S1p-M, Bacmid-S1 M13 universal primer qualification result electrophoretogram; 1,2: restructuring empty vector control M13 universal primer qualification result; 3. negative control; 4. recombinant vector Bacmid-S1p-M; 5. recombinant vector Bacmid-S1; M.250bpDNAMarker.
Fig. 3 .Western-blot identifies the expression of S1 albumen, S1p albumen, M albumen; 1. recombinant baculovirus Bv-S1p-M expression product; 2,3: recombinant baculovirus Bv-S1 expression product; 4: be negative control group (not containing the wild-type baculovirus product of genes of interest); M: pre-dyed protein molecular weight standard.
Detailed description of the invention
The present invention is further described below in conjunction with Figure of description and specific embodiment.Unless stated otherwise, the present invention adopts reagent, equipment are the art conventional reagent and equipment.
Embodiment 1
The structure of recombinant vector Bacmid-S1p-M, Bacmid-S1
The acquisition of 1.PEDVS1 gene, S1 incomplete antigen gene and M gene: the gene order according to the new isolated strain of PEDV designs a pair Auele Specific Primer, and primer sequence is as follows:
S1-P1：5'CGGAATTCCAAGATGTCACTAGGTGCCAGTCTA3'
S1-P2：5'CCCTCGAGCTATCTAATACTCATACTAAAGTTGGTGGG3'
M-P1：5'GAAGGCCTATGCATCACCATCACCATCACGATTA3'
M-P2：5'CCAAGCTT
TTAGACTAAATGAAGCACTTTCTC3'
S1p-P1:5'CCCTCGAG
ATGGTACTTCTAACACTTAGCCTACCAC3'
S1p-P2:5'CATGCATGC
CTAAGGATCTGAGGAATTACTGCAAACA3'
With the PEDV pathological material of disease of the positive, extract its total serum IgE, respectively with each downstream primer P2 for Auele Specific Primer makes RT; Then with cDNA product for template, application round pcr amplifies S1 full genome, S1p(S1 Gene Partial epitope) and M gene; Wherein the nucleotide sequence of the encoding gene S1p of the part S1 albumen of Porcine epidemic diarrhea virus is as shown in SEQIDNO:1; The nucleotide sequence of S1 full genome is as shown in SEQIDNO:2; The nucleotide sequence of M gene is as shown in SEQIDNO:3.
2. the structure of recombinant transfer vector pFastbac-Dual-S1p-M, pFastbac-HTB-S1: carry out in 0.5mL centrifuge tube: transfer vector pFastbac-Dual, pFastbac-HTB and S1p, M, S1 gene etc. are carried out double digestion simultaneously, product after double digestion is reclaimed, recovery product being connected, transformed simultaneously, extracting being accredited as positive plasmid after PCR or the checking of double digestion (see figure 1).
3. the structure of recombinant shuttle vector Bacmid-S1p-M, Bacmid-S1 and acquisition: take out DH10Bac competent cell, after ice bath melted, add 10 μ L transfer vector pFastbac-Dual-S1p-M, pFastbacHTB-S1 respectively, mix gently, ice bath 30min is 42 DEG C of heat stress 90sec then, then ice bath effect 5min; Add the fresh LB fluid medium of 800 μ L37 DEG C preheatings, 37 DEG C of 150r/min are centrifugal after cultivating 1h discards most of supernatant, and remainder about 80 μ L liquid, is layered on an anti-flat board of LB tri-after resuspended, flat board is put into 37 incubators and is inverted and cultivates 48h; Choose 10 white clones, again rule at the anti-flat board of fresh LB tri-.37 DEG C of incubated overnight.White monoclonal selected by the flat board that Bluo-Gal and IPTG rule again prove dominant comprising, picking pure white bacterium colony is in LB tri-resistant to liquids, and 37 DEG C of 150r/min cultivate 16 ~ 20h;
4. the qualification of recombiant plasmid Bacmid-S1p-M, Bacmid-S1: comprise the positive and negative primer sites of M13 in shuttle vector, be positioned at both sides, LacZaattTn7 site, may be used for PCR inspection, the agarose gel electrophoresis qualification of use 0.8% after PCR completes, qualification result as shown in Figure 2.
Embodiment 2
The acquisition of recombinant baculovirus Bacmid-S1p-M, Bacmid-S1
The recovery of 1.1Sf9 insect cell and Secondary Culture
2. recombinate Bacmid-S1p-M, Bacmid-S1 plasmid transfection Sf9 cell by 2 × 10
5density inoculates Sf9 cell on the Tissue Culture Plate in 12 holes, carries out transfection, discard old cell culture fluid when waiting cell to grow to 80 ~ 90% full scale, changes into not containing the Grace cell culture fluid of serum; Following solutions is prepared: solution A: with the incomplete cell culture fluid of GRACE, 1.6 μ gDNA are diluted to 100 μ L in aseptic 1.5mL centrifuge tube; Solution B: dilute 4.0 μ L liposome Lipofectanmine2000 to 100 μ L with the incomplete cell culture fluid of GRACE; Mixed solution A and solution B, room temperature leaves standstill 20min to form the complex of liposome and DNA; The complex of liposome and DNA is slowly dropwise added cell surface (not discarding nutritional solution); The cell culture fluid containing transfection liquid is changed into cell culture fluid containing 10% hyclone after 4 ~ 6h; After cell covers with monolayer, growth-promoting media is changed into the GRACE cell culture fluid of 2% hyclone; Observation of cell state, stop cultivating to when there is cytopathy, collect viral passages, the recombinant baculovirus Bacmid-S1p-M obtained and recombinant baculovirus Bacmid-S1 is identified the expression of S1 albumen, S1p albumen, M albumen by Western-blot, the results are shown in Figure 3, as shown in Figure 3, S1 gene, M gene and S1p gene can utilize virus expression systems high level expression.
The preparation of embodiment 3 vaccine:
After obtaining recombinant baculovirus Bacmid-S1p-M and recombinant baculovirus Bacmid-S1 by embodiment 2, virus expression systems is utilized to express M albumen and S1 albumen.Described step is for carry out amplification culture by recombinant baculovirus Bv-S1p-M or recombinant baculovirus Bv-S1, and purification reclaims and obtains S1 albumen or S1p albumen and M albumen; Amplification culture is: after obtaining the recombinant baculovirus of appropriate titer, and in cell is in, during logarithmic (log) phase, (density is 1 ~ 2 × 10
6cell/mL) connect malicious infection cell, use serum-free condition to cultivate insect cell, contribute to the purification of later stage destination protein like this.But generally should according to needs, should the phase adds FBS or BSA of 0.1 ~ 0.5% to protect recombiant protein not to be hydrolyzed after infection.After cell inoculation recombinant virus under 28 DEG C of conditions, cultivate 72h and start to gather in the crops metainfective cell.
Described purification reclaims the needs comprised the steps: in order to later stage purifying protein, with the addition of 6 histidine during contrived experiment at 5 ends of two sections of genes of interest, uses affinity chromatography medium Ni-NTA to carry out purification recovery.Recycling step is as follows: after (1) cell connects poison, and cultivate 72 hours for 27 DEG C, then collect sick cell, every 100mg cell (weight in wet base) adds 1 ~ 5mL lysate (having added 10 μ LPMSF in every 1mL extraction agent), ultrasonic degradation cell.A large amount of heat production that continuous ultrasound causes should be avoided, can the short time be divided into, repeated ultrasonic, avoid solution overheated by certain interval time.Finally become clear with liquid.(2) 10000rpm, 4 DEG C centrifugal 3 minutes, collects the soluble protein in supernatant.(3) with BindingBuffer by the liquid equimultiple dilution back loading upper prop after sick cell cracking, flow velocity be 10 times of column volumes/hour, collect stream and wear liquid.(4) use the BindingBuffer of 15 times of column volumes to rinse pillar, wash away foreign protein.(5) use ElutionBuffer eluting, collect eluting peak.Attention: eluting peak can be in charge of collection, every 1ml collects 1 pipe, and adopts albumen monitor to monitor, and collects eluting peak.(6) after eluting, use the deionized water wash pillar of 10 times of column volumes successively, then balance (ethanol will by filler submergence), 4 DEG C of preservations with 20% ethanol of 3 times of column volumes.
Destination protein is after purification reclaims, with 0.1% formalin-inactivated 12 hours, add adjuvant (adjuvant concentration remains on 20%) after deactivation respectively fully to mix afterwards, in order to can reduce vaccine to laboratory animal produce stress, in the vaccine prepared, add sodium thiosulfate (Na
2s
2o
3) keep final concentration be 5%, vaccine be put in after having prepared 4 DEG C for subsequent use.
Embodiment 4
Zoopery:
Choose that 24 outward appearances are normal, the breeding same period, the sow heavy in pig that the antibody tests such as PEDV, TGEV and RV are all negative, is divided into 4 large groups: wherein experimental group 1 and experimental group 2 are respectively divided into two groups, every group 3 sows at random.Matched group is divided into contrast 1, matched group 2, matched group 3 and matched group 4, often organizes 3.Immunity: after prepared by vaccine, passive immunity 2 times, 2 weeks, interval between initial immunity and booster immunization.Collect the colostrum of every sow immediately after farrowing and gather blood, detecting antibody titer respectively by ELISA kit.By immune group, respectively to the suckling colostrum piglet of 3 days homology Porcine epidemic diarrhea virus counteracting toxic substances, observe counteracting toxic substances protected effect and record.Sow immunization experiment grouping situation is as table 1.
Table 1 sow immunization experiment grouping situation
Experimental result: sow head exempts from booster immunization after 2 weeks, detect through ELISA kit, experimental group 1, experimental group 2, matched group 1, matched group 3 and matched group 4 etc. five groups experiment sow all can produce specific antibody, wherein experimental group 1, experimental group 2, matched group 1 can detect higher antibody horizontal in first Ruzhong and matched group 3 and matched group 4 only can detect a small amount of antibody in first Ruzhong.Matched group 2 there is no specific antibody and produces.The antibody titer that the subunit oil emulsion adjuvant vaccine of experimental group 1 and experimental group 2 and aluminium glue Adjuvanted vaccines produce is suitable, and is better than the inactivated vaccine of matched group 1 and the vaccine of matched group 3,4 prokaryotic expression.
Table 2 piglet counteracting toxic substances Protection result
Before Porcine epidemic diarrhea virus counteracting toxic substances, each group piglet all occurs without symptom of diarrhea, and spirit is normal with appetite, and growth conditions is good; After counteracting toxic substances, all there is symptom of diarrhea two days later at counteracting toxic substances in the piglet of matched group 2, and last from days, appetite and spirit are all poor, occur watery diarrhea and occur dewatering symptom until occur dead when sick pig is serious.Matched group 1 after pig counteracting toxic substances of farrowing, have 8 piglets to occur symptom of diarrhea, all the other piglets are all normal.In experimental group 1, piglet has 1 to occur symptom of diarrhea, and in order, appetite is normal for all the other piglet growths.In experimental group 2, oily adjuvant, aluminium glue adjuvant group all do not have piglet to occur diarrhoea status.
Total upper result of the test shows; the specific antibody of higher level can be produced after sow Pigs Inoculated epidemic diarrhea recombinant baculovirus Bacmid-S1p-M, Bacmid-S1 subunit vaccine; newborn piglet is searched for food after colostrum can obtain good antibody Vaccine effectiveness; greatly reducing newborn piglet and infect PEDV probability, effectively reducing the mortality rate of piglet because causing after infection PEDV.PEDVS1 and the M immune protein that baculovirus expression system can be expressed efficiently, thus significantly improve the content of Effective Antigens in subunit vaccine, and lower than traditional inactivated vaccine on production cost.Therefore, for new generation porcine epizootic diarrhea subunit vaccine provides direction.
SEQUENCELISTING
<110> Agricultural University Of South China
<120> porcine epizootic diarrhea recombinant baculovirus genetic engineering subunit vaccine and preparation method thereof and application
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acgctaacactccttagtggtacattgcttgtagagggctataaggttgctactggcgta480
caggtaagtcaattgcctgatttcgtcacagtcgccaaggccactacaacaattgtctat540
ggacgtgttggtcgttcagtcaatgcttcatctggcactggttgggctttctatgtccgg600
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aaagtgcttcatttagtctaa681
<210>4
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<213>S1-P1
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<212>DNA
<213>M-P2
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<213>S1p-P1
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Claims (4)
1. a preparation method for porcine epizootic diarrhea recombinant baculovirus genetic engineering subunit vaccine, is characterized in that, comprises the steps:
S1. the encoding gene S1p of the part S1 albumen of Porcine epidemic diarrhea virus and the encoding gene of entire M protein are cloned in baculovirus expression system and obtain recombinant baculovirus Bv-S1p-M;
S2. recombinant baculovirus is carried out amplification culture, purification reclaims and obtains S1p albumen and M albumen;
S3. can obtain after S1p albumen and M albumen and pharmaceutically acceptable adjuvant fully being mixed and organize Baculovirus Gene engineering subunit vaccine;
Wherein the nucleotide sequence of the encoding gene S1p of the part S1 albumen of Porcine epidemic diarrhea virus is as shown in SEQIDNO:1;
Adjuvant described in S3 is oil emulsion adjuvant;
Described in S1, the construction method of recombinant baculovirus Bv-S1p-M is: the encoding gene S1p of the part S1 albumen of Porcine epidemic diarrhea virus and the encoding gene of entire M protein are cloned in baculovirus transfer vector pFastbac-Dual, obtain recombinant vector pFastbac-Dual-S1p-M; Recombinant vector pFastbac-Dual-S1p-M is transformed into pre-containing in the DH10Bac competent cell of baculovirus skeleton plasmid, and by extracting recombinant vector after homologous recombination, recombinant vector again transfection Sf9 cell produces recombinant baculovirus.
2. the preparation method of vaccine according to claim 1, it is characterized in that, amplification culture described in S2 is: be 1 ~ 2 × 10 by density
6after the recombinant baculovirus of cell/mL connects malicious infected insect cell, insect cell containing 0.1 ~ 0.5% FBS or BSA serum-free medium in, cultivate 72h for 28 DEG C.
3. the preparation method of vaccine according to claim 1, it is characterized in that, described in S2, purification is recovered as: the insect cell after centrifugal recovery S2 amplification culture, utilizes affinity chromatography medium Ni-NTA to carry out purification and reclaim destination protein.
4. the porcine epizootic diarrhea recombinant baculovirus genetic engineering subunit vaccine for preparing of method described in any one of claims 1 to 3.
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