CN102559614A - Classical swine fever vaccine and preparation method thereof - Google Patents

Abstract

The invention relates to a classical swine fever vaccine and a preparation method thereof and belongs to the technical field of biomedicine. According to the invention, main antigen genes E2 and E0 of the classical swine fever virus are displayed on the surface of silkworm rhabdovirus bursa by constructing a recombinant rhabdovirus and utilizing a rhabdovirus surface display technology; the recombinant rhabdovirus is produced by using silkworm pupae as a bioreactor; and the classical swine fever vaccine is produced by using the recombinant rhabdovirus as classical swine fever vaccine source. Compared with the traditional production method for the classical swine fever vaccine, the method has the characteristics of high safety, low production cost, high yield, simplicity in operation and suitability for large-scale production and has wide application prospect.

Description

A kind of swine Fever Vaccine and preparation method thereof

Technical field

The present invention relates to a kind of swine Fever Vaccine and preparation method thereof, relate in particular to the preparation of recombinant plasmid and recombinant virus in the swine Fever Vaccine preparation, belong to the biological medicine technology field.

Background technology

(hog cholera HC) claims that again (classical swine fever CSF), is the hot transmissible disease of a kind of height contact that is caused by CSFV (CSFV) to classic swine fever, and mortality ratio is up to more than 90% to swine fever.1984 OIE (OIE) list it in category-A transmissible disease, also be one of main transmissible disease of paying close attention to of the World Food Programme and national governments.

After Hungary HutyraK-oves processed the swine fever hyper-immune serum in 1908, people used hyper-immune serum-blood poison intramuscular injection simultaneously to come immune swinery always.But because hyper-immune serum costs an arm and a leg, obtain difficulty, the blood poison has the danger of the poison that looses in addition, thus the various countries scholar afterwards 60 in the period of develop many inactivated vaccines in succession, wherein Superlysoform and Viola crystallina inactivated vaccine once were widely used.Though inactivated vaccine safety, preservation period is short.

Nineteen fifty-five, people such as Zhou Taichong successfully prepare the rabbitization low virulent strain with CSFV crossdrift virulent strain after the rabbit body goes down to posterity for 214 times.Continue to go down to posterity 480 times, become commercial standard vaccine strain.Be named as Chinese vaccine strain (C-strain) or hog cholera lapinised virus (HCLV) strain afterwards, a lot of commercial vaccine strains are all derived by it.Be widely used in world many countries since nineteen fifty-seven, be most popular in the world at present a little less than malicious seedling, played keying action to control CSF is popular.Compare other commercial vaccine, its security is higher, and bacterium continues to exist in the pig body to be no more than for 3 weeks, and can not cause the morbidity of piglet and farrowing sow; Immune effect is better, and immunity once just can produce protection completely, and guard time reaches 1 year even be lifelong.Although conventional less toxic vaccine is safe and effective; But there is immune antibody and infects the undistinguishable problem of antibody; Can not detect a wild virus infection in the immune swinery through serological method, this anti-system for swine fever is very unfavorable with purification, also just because of this reason; Take the pig of hog cholera immune policy country and the outlet of goods thereof to receive strict restriction, this just makes exploitation to become the task of top priority with the new generation vaccine that serological method is distinguished immune swine and infected pigs.

Subunit vaccine is one type of ability is distinguished immune animal and infection animal easily through serological method a vaccine.At present existing 2 kinds of subunit vaccines have obtained registration in European Union, and these 2 kinds of vaccines all are that CSFV E2 albumen with baculovirus expression is as immunogen.But also there are some defectives in this vaccine, and test shows that the E2 protein subunit vaccine could produce the protection to wild virus infection behind immune 14d; Simultaneously; If there is low-level maternal antibody to exist during immunity in the body; At least want immunity just can induce clinical protection twice, even so, most of pigs that produced clinical protection still have heating, oligoleukocythemia and viremia when attacking poison situation occurs.In addition, can the E2 protein subunit vaccine prevent horizontal transmission and vertical transmission also to remain to be confirmed.

The live recombinant vectors vaccine is the marker vaccine that begins one's study at first, and live vector vaccine can cause humoral immunization and cellular immunization, and can duplicate in vivo, can induce long-term and immunoprotection efficiently.The virus vector that is used for the swine fever marker vaccine has poxvirus, pseudorabies virus (PRV), porcine adenovirus (PAV) etc.But they all have obvious defects: vaccinia virus recombinant possibly have the potential infectivity to the people who did not inoculate bovine vaccine; Vaccinia virus recombinant propagates in the environment and possibly recombinate with the wild-type vaccinia virus, virulence occurs and returns strong phenomenon; Vaccinia virus is lower to the infectivity of pig, must inject the vaccinia virus recombinant of high dosage and just can induce effective protection; Be not suitable for the popular area of pseudoabies based on the PRV carrier bacterin, because PRV antibody has certain influence for immunity and serology detection; Though and rPAV-E2 and rPRV-E2 are efficient and safety, do not obtain concrete vaccine test or field experiment and confirm.

Dna vaccination possesses a lot of advantages; Develop very fast; But also there is the potential potential safety hazard; Whether can be incorporated in the host genome, whether can cause that problem such as immunological tolerance it be unclear that like DNA, and have certain potential safety hazard, the dna vaccination immune efficacy still remains further to be improved in addition.

Based on above various situation, studying novel, effective swine Fever Vaccine has been investigator's striving direction, and E2, E0 albumen be the major antigen site of CSFV, is the main target spot of swine Fever Vaccine development.

The baculovirus surface display system is a kind of new eucaryon display systems that grew up in recent years; Can remedy the deficiency of protokaryon display systems; And the advantage of maintenance surface display system; Promptly be convenient to the purifying of foreign protein through the recombinant baculovirus particle of screening expression differential protein; Can be used to show to need the just active complicated eukaryotic protein of the function of appeal of posttranslational modifications such as glycosylation, disulfide linkage isomerizing, have broad application prospects at aspects such as the screening of high-affinity antibody and polypeptide drugs, proteantigen epitope analysis.

Summary of the invention

In view of this, the object of the present invention is to provide a kind of swine Fever Vaccine that overcomes above-mentioned defective.The present invention is through making up a kind of recombinant silkworm baculovirus; And use baculovirus surface display technology that major antigen gene E2, the E0 fusion of CSFV are illustrated in silkworm baculovirus cyst membrane surface; Produce recombinant baculovirus with silkworm pupa as bio-reactor; And produce as swine Fever Vaccine is former with this recombinant baculovirus, have that security is good, production cost is low, output is high, easy handling, be applicable to the characteristics of scale operation than traditional swine Fever Vaccine production.

For achieving the above object, the technical scheme that the present invention adopts is following:

A kind of silkworm with recombinant baculovirus Bmgp64-E2-2A-E0; This virus is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on December 15th, 2011; It abbreviates CGMCC (address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City as; Institute of Microorganism, Academia Sinica, postcode 100101) preservation, classification called after Bombyx mori nuclear polyhydrosis virus ( Bombyx mori nucleopolyhedrovirus), deposit number is CGMCC No.5604.

The preparation method of above-mentioned silkworm with recombinant baculovirus may further comprise the steps:

(a) method by pcr amplification obtains baculovirus envelope protein gp64 signal peptide gene sequence SP and baculovirus envelope protein gp64 membrane-spanning domain gene order TM, through BamH I/ EcoR I with XhoI/ HindThe III double digestion inserts pFastBacI carrier MCS upstream and downstream two ends, makes up surface display system transfer vector pFastBacI-gp64;

(b) adopting the pvL1393-E2-2A-E0 recombinant plasmid is template, is that primer carries out pcr amplification with SEQ ID:NO.6 and SEQ ID:NO.7, gene fusion construct E2-2A-E0;

(c) on fusion gene E2-2A-E0 double digestion product cloning to step (a) the gained surface display system transfer vector pFastBacI-gp64 with step (b) gained, and screening positive clone, obtain to shift recombinant plasmid pFastBacI-gp64-E2-2A-E0;

(d) step (c) gained is shifted recombinant plasmid pFastBacI-gp64-E2-2A-E0 transformed into escherichia coli DH10Bac competent cell; Carry out the screening of blue hickie on the LB culture plate of kantlex, qingfengmeisu qiong, tsiklomitsin, X-gal and IPTG containing; Picking hickie behind the lucifuge cultivation 40-48h, bacterium liquid dilution 10 behind the cultivation 16-20h ⁶Again coated plate is done screening, after picking hickie to LB substratum shakes bacterium cultivation 16-20h, with Virahol extracting recombinant baculovirus genome, and is PCR with the M13 universal primer and identifies;

(e) get step (d) the successful recombinant virus genomes of identifying through liposome mediated-method transfection silkworm BmN cell; The morbidity back obtains zero for viral suspension 0-4 ℃ preservation; Extract viral genome and carry out the PCR evaluation, obtain said silkworm with recombinant baculovirus Bmgp64-E2-2A-E0 with the M13 universal primer.

Above-mentioned recombinant baculovirus Bmgp64-E2-2A-E0 expressed proteins, its aminoacid sequence is shown in SEQ ID:NO.11.

Above-mentioned proteic preparation method may further comprise the steps:

(a) above-mentioned recombinant baculovirus Bmgp64-E2-2A-E0 infected silkworm BmN cell is carried out virus amplification;

(b) with stab inoculation inoculation silkworm five-age larva, pupa;

(c) collect the above-mentioned albumen of expressing.

The present invention further provides above-mentioned silkworm with recombinant baculovirus Bmgp64-E2-2A-E0 application in the swine Fever Vaccine preparation.

The present invention also provides the application of above-mentioned albumen in the swine Fever Vaccine preparation.

The present invention has following beneficial effect:

The present invention utilizes silkworm pupa, five-age larva to be bio-reactor; Efficiently express CSFV major antigen albumen E2 and E0; Through with baculovirus gp64 proteic N end signal peptide and C end span film district and E2-2A-E0 gene fusion; Make CSFV major antigen recombination fusion protein be showed in baculovirus cyst membrane surface and form the pseudo-virus of baculovirus that has the exogenous virus major antigen; This puppet virus has the immunogenicity of exogenous virus, can be used as the recombination engineered vaccine, and baculovirus expression vector system belongs to eukaryotic expression system; Having the processing rhetorical function after the translation, is that the foreign protein of expressing has and more approaches natural biological activity.Because albumen is nontoxic to people and animals in the silkworm pupa, so the recombinant vaccine that the present invention is bio-reactor with the silkworm chrysalis to produce is little to the spinoff that people and animals produce, and is applicable to scale operation, production cost is low.In addition, the present invention's development of using silkworm baculovirus surface display technological expression CSFV recombination fusion protein E2-2A-E0 to be used for swine Fever Vaccine also still belongs to the first.

Description of drawings

Fig. 1 is the collection of illustrative plates of the baculovirus surface display system transfer vector pFastBacI-gp64 that makes up among the present invention;

Fig. 2 is the collection of illustrative plates synoptic diagram of the recombinant transfer plasmid pFastBacI-gp64-E2-2A-E0 that makes up of the present invention;

Fig. 3 is the mode chart of the recombinant baculovirus Bmgp64-E2-2A-E0 that makes up of the present invention.

Embodiment

Be noted that following specifying all is exemplary, being intended to provides further invention to the present invention.Except as otherwise noted, all Science and Technology terms of using of this paper have with the present invention under the identical meanings of person skilled common sense.

Specify particular content of the present invention below in conjunction with embodiment.

Embodiment 1: the acquisition of fusion gene E2-2A-E0

The sequence (gene accession number: E0 is EF015292.1, and E2 is DQ907718.1) that goes up CSFV E2, E0 gene according to NCBI designs the upstream primer SEQ ID:NO.6 of raq gene and the downstream primer SEQ ID:NO.7 of E0 gene, and upstream primer is introduced EcoRThe I restriction enzyme site, downstream primer is introduced XhoThe I restriction enzyme site is a template with the pvL1393-E2-2A-E0 recombinant plasmid that the Zhang Zhi of Chinese Academy of Agricultural Sciences virtue teacher gives, and carries out pcr amplification, obtains fusion gene E2-2A-E0, its nucleotide sequence and aminoacid sequence such as SEQ ID:NO.10.Specific procedure and reaction system are following:

Response procedures:

Reaction system:

10×PCR Buffer 5 μl

25mM MgCl ₂ 4 μl

2.5mM dNTPs 4 μl

SEQ ID: NO.6 1 μl

SEQ ID: NO.7 1 μl

Template 1 μ l

KOD Plus DNA polysaccharase (TOYOBO company) 1 μ l

Add aseptic ddH ₂O to 50 μ l

The PCR product is used EcoR I, XhoAfter I (all available from Invitrogen company) carried out double digestion, rubber tapping was reclaimed for future use.

Embodiment 2: the structure of baculovirus surface display system transfer vector pFastBacI-gp64

(the gene number of landing is: L33180.1) to go up the baculovirus gp64 gene order that obtains according to NCBI; Design primer SEQ ID:NO.2, SEQ ID:NO.3 go out its signal peptide (SP) sequence through overlap extension pcr (SOE-PCR) specific amplified; With SEQ ID:NO.4, SEQ ID:NO.5 is primer; The wild baculovirus genome that extracts goes out to stride diaphragm area (TM) sequence for the template specific amplified; Like SEQ ID:NO.9, and be that stencil design is identified upstream primer SEQ ID:NO.1 with the SP sequence of obtaining on the NCBI, it is following that each goes on foot concrete response procedures:

(1) the SP sequence is synthetic:

Response procedures:

Reaction system:

10×PCR Buffer 5 μl

25mM MgCl ₂ 4 μl

2.5mM dNTPs 4 μl

SEQ ID: NO.2 1 μl

SEQ ID: NO.3 1 μl

KOD Plus DNA polysaccharase (TOYOBO company) 1 μ l

Add aseptic ddH ₂O to 50 μ l

The PCR product is used BamHI, EcoAfter R I (all available from Fermentas company) carried out double digestion, rubber tapping was reclaimed for future use.

(2) the TM sequence is synthetic:

Response procedures:

Reaction system:

10×PCR Buffer 5 μl

25mM MgCl ₂ 4 μl

2.5mM dNTPs 4 μl

SEQ ID: NO.4 1 μl

SEQ ID: NO.5 1 μl

Genomic templates 1 μ l

KOD Plus DNA polysaccharase (TOYOBO company) 1 μ l

Add aseptic ddH ₂O to 50 μ l

The PCR product is used XhoI, HindAfter III carried out double digestion, rubber tapping was reclaimed for future use.

With the PCR product behind (1), (2) the two groups of double digestions; Be cloned into respectively successively on the unloaded plasmid of pFastBacI (available from Invitrogen company); Transformed into escherichia coli TG1 competent cell (available from Invitrogen company); Picking list bacterium colony utilizes above-mentioned PCR primers designed SEQ ID:NO.1 and SEQ ID:NO.5 to carry out PCR and identifies, and carries out double digestion and identify with order-checking; The plasmid that positive colony is got in screening is baculovirus surface display system transfer vector pFastBacI-gp64, and is as shown in Figure 1.

Embodiment 3: the structure of baculovirus surface display system transferring plasmid pFastBacI-gp64-E2-2A-E0

To transfer vector pFastBacI-gp64, screening positive clone is baculovirus surface display system transferring plasmid pFastBacI-gp64-E2-2A-E0, and is as shown in Figure 2 with the fusion gene E2-2A-E0 double digestion product cloning that reclaims among the embodiment 1.

Embodiment 4: recombinant baculovirus Bmgp64-E2-2A-E0 obtaining and identifying

With the recombinant transfer plasmid pFastBacI-gp64-E2-2A-E0 transformed into escherichia coli DH10Bac competent cell (available from Invitrogen company) that is obtained among the embodiment 3; On the LB solid culture plate (ordinary method preparation) that contains kantlex, qingfengmeisu qiong, tsiklomitsin, X-gal and IPTG (sec.-propyl-β-D-sulfo-galactopyranoside), carry out blue hickie screening available from Bio Basic Inc company; Picking list bacterium colony hickie behind the lucifuge cultivation 48h, bacterium liquid dilution 10 behind the cultivation 16h ⁶Again coated plate is done screening, after picking hickie to LB substratum shakes bacterium cultivation 16h, with Virahol extracting recombinant baculovirus genome in super clean bench, identifies with the M13 universal primer PCR whether swivel base is successful.

M13 upstream primer: 5 '-GTTTTCCCAGTCACGAC-3 '

M13 downstream primer: 5 '-CAGGAAACAGCTATGAC-3 '

With identifying the method for successful recombinant baculovirus genome with reference to the lipofectamine Cellfectin of Invitrogen company II Reagent specification sheets; Through liposome mediated-method transfection silkworm BmN cell (is first generation cross-fertilize seed cyanines pine * bright moons; Biochemical institute gives by Shanghai); Treat this silkworm BmN cell morbidity (microscopically observations) back collection first-generation recombinant virus suspension and in 4 ℃ of preservations; Extract viral genome with Viral DNA Kit (200) (available from OMEGA company), carry out PCR with the M13 universal primer then and identify.Gained is identified successful recombinant baculovirus called after Bmgp64-E2-2A-E0, as shown in Figure 3.

Embodiment 5: expression (amplification) and the evaluation of recombinant baculovirus Bmgp64-E2-2A-E0 in silkworm BmN cell

In the dosage infected silkworm BmN cell log vegetative period silkworm BmN cell of the recombinant baculovirus Bmgp64-E2-2A-E0 that obtains among the embodiment 4 with MOI=0.1pfu/cell; In containing 1 * Sf-900 II SFM substratum (available from Invitrogen company) of 10% foetal calf serum, cultivate 3～4 d for 27 ℃, collect culture supernatant; 4 ℃, it is frozen in-70 ℃ that centrifugal 10 min of 500 * g, supernatant store in 4 ℃ or packing.Recombinant baculovirus after the amplification with MOI=10 pfu/cell dosage infected silkworm BmN cell (be first generation cross-fertilize seed cyanines pine * bright moons, biochemical institute gives by Shanghai), is cultivated 72 h at 27 ℃, 4 ℃, the centrifugal 10min collecting cell deposition of 2000g; This cell precipitation is resuspended with the PBS washing of pH 7.4, and sample mixes with 2 * SDS sample-loading buffer respectively, 60 ℃ of heating 30 min; Carry out the SDS-PAGE protein electrophoresis; Western blotting analyzes, and the result shows that fusion rotein E2-2A-E0 is successful expression in silkworm BmN cell.

Embodiment 6: expression and the evaluation of recombinant baculovirus Bmgp64-E2-2A-E0 in silkworm five-age larva and silkworm chrysalis

(is first generation cross-fertilize seed cyanines pine * bright moons with the silkworm five-age larva of the recombinant baculovirus Bmgp64-E2-2A-E0 after embodiment 5 amplification after with the dosage belly joint second from the bottom place percutaneous puncture-inoculation molting of MOI=10 pfu/cell with the silkworm chrysalis of pupating two days; Zhongqi Biological Pharmaceutical Co., Ltd., Zhejiang); Typical virus infection symptom appears in the 5th day most of larva and silkworm chrysalis; Cut the larva abdominal foot and collect the silkworm hemolymph; Add 0.5% (w/v) inhibitor 8-hydroxy-quinoline, the centrifugal 10min of 12000rpm gets supernatant; With silkworm chrysalis-80 ℃ storage.The homogenate of getting cleer and peaceful morbidity silkworm chrysalis on the silkworm hemolymph of 12000rpm after centrifugal adds isopyknic 2 * protein sample-loading buffer (100Mm Tris-HCl respectively; 4% SDS; 0.1% tetrabromophenol sulfonphthalein, 10% glycerine), 60 ℃ of heating 30min; Get 20 μ l and carry out the SDS-PAGE protein electrophoresis, and carry out Western blotting and analyze.The result shows that fusion rotein E2-2A-E0 has obtained successful expression in silkworm larva and silkworm pupa.

Embodiment 7: the acquisition of novel swine Fever Vaccine

(1) separation and purification recombinant virus Bmgp64-E2-2A-E0 from silkworm chrysalis:

A. get the silkworm chrysalis sample that 300g infects through Bmgp64-E2-2A-E0, after thawing under 4 ℃, with 4 ℃ of ddH that place 1 h ₂O 300ml mixes, and places refiner, and homogenate 2min places 2min on ice, repeats 9 times;

B. homogenate is added in the 1L Centrifuge Cup, trim, the centrifugal 60min of 6000rpm gets supernatant, carefully discards grease;

C. the 6000rpm centrifuged supernatant is poured in another 1L Centrifuge Cup, trim, the centrifugal 60min of 6000rpm gets supernatant, abandons grease;

D. repeat c once;

E. 6000rpm centrifugal supernatant is poured the 50ml centrifuge tube into, trim, and the centrifugal 30min of 12000rpm gets supernatant, abandons grease (it is inferior to repeat this step 3);

F. get the supernatant 300ml of 12000rpm after centrifugal; Carry out ultrafiltration with 300kD hollow-fibre membrane film bag ultrafiltration system 4 ℃ (available from Sartorius companies); Be appearance ultrafiltration on the volume ratio of 1:1 according to supernatant and PBS, so repeat final trapped fluid 100ml 10 times; Clean the film bag with 200ml PBS again, and merge with the liquid that dams;

G. the sample after the ultrafiltration is sub-packed in the ultra in pipe of sterilization, balance, the centrifugal 40min of 40000rpm ultra-high speed on super clean bench;

H. 40000rpm centrifugal after, get all supernatants, and the resuspended liquid of deposition (all depositions are resuspended with 50ml PBS) carries out band centrifugation respectively, last kind in proper order as follows: PBS 250ml; Sample 300ml, 30% sucrose solution 400ml, 55% sucrose solution is full of, and sample introduction adds upper cap after finishing; Close door, the open vacuum pump begins centrifugal, and centrifugal end back is with following kind of 56% sucrose solution; With the monitoring of nucleic acid-protein detector, collect and flow out sample, altogether 150mL;

I. sample leaves standstill deactivation 24h for 37 ℃ with thousand/dicarbaldehyde behind the band centrifugation;

J. ultrafiltration desugar: sample to 50mL, is diluted to 100mL with sterilized water with the ultrafiltration of 300kD hollow-fibre membrane film bag again after the deactivation, and ultrafiltration is to 50mL again; So repeat 10 times, finally holding back volume is 50mL, uses the aseptic moisture of 300mL 3 times again; Each 100mL; The circulation flushing ultrafiltration system finally is concentrated into 50mL, merges the two times of ultrafiltration liquid that dams;

(2) freeze-drying: sample after the ultrafiltration desugar, active with the indirect elisa method detection of biological, activated sample carries out packing, in-40 ℃ of lyophilizes;

(3) preserve and detect:

Novel swine Fever Vaccine lyophilized powder is processed in aseptic subpackaged warp-40 ℃ lyophilize.Simultaneously, identify the biological activity of fusion rotein E2-2A-E0 in the lyophilized powder again with ELISA.The 12%SDS-PAGE electrophoresis, test sample has the expression of target protein at the 95kD place.

(4) the Balb/c mouse in 4 ages in week of recombinant virus particle immunity after getting purifying and identifying prepares polyclonal antibody, does serum neutralization test, and neutralization is tired and can be reached 1:32, shows to have good immunogenicity.The serum neutralization test step is following:

The Balb/c mouse polyclonal antibody of preparation is handled 30 min in 56 ℃ of water-baths, use the serial dilution of serum-free Sf-900TMSFM (1X) substratum (available from Invitrogen company) difference multiple proportions then to 2-8;

Getting the different dilution serum with equal volume of recombinant baculovirus Bmgp64-E2-2A-E0 suspension that 50 μ L prepare in advance mixes; Hatch 1h for 37 ℃; Balb/c mice serum test group with recombinant baculovirus Bmgp64-E2-2A-E0 immunity; With the Balb/c mice serum negative control group of PBS immunity, carry out in the serum and experiment;

Get the good silkworm BmN cell of growth conditions, diluting cells density to 1 * 105/mL, 500 μ L/ holes add in 24 orifice plates;

With 100 μ L/ holes inoculation BmN cell, different extent of dilution serum are done three repetitions, 27 ℃ of cultivations in the cell culture incubator with virus-serum mixture;

Observation of cell pathology situation, the calculating antibody neutralization is tired.

The above is merely preferred embodiment of the present invention, is not the present invention is done any pro forma restriction; Though the present invention discloses as above with preferred embodiment; Yet be not that any those of ordinary skill in the art is in the scope that does not break away from spirit of the present invention in order to qualification the present invention; Any modification of being done, equivalence variation and modification etc. all still belong within protection scope of the present invention.

SEQUENCE LISTING

< 110>Tianjin Yaoyu Biotechnology Co., Ltd.

< 120>a kind of swine Fever Vaccine and preparation method thereof

<130> 111665-I-CP-TJYU

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<170> PatentIn version 3.5

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Met Arg Leu Ala Cys Lys Glu Asp Tyr Arg Tyr Ala Ile Ser Ser Thr

1 5 10 15

Asp Glu Ile Gly Leu Leu Gly Ala Gly Gly Leu Thr Thr Thr Trp Lys

20 25 30

Glu Tyr Asn His Asp Leu Gln Leu Asn Gly Gly Thr Val Lys Ala Ser

35 40 45

Cys Val Ala Gly Ser Phe Lys Val Thr Ala Leu Asn Val Val Ser Arg

50 55 60

Arg Tyr Leu Ala Ser Leu His Lys Lys Ala Leu Pro Thr Ser Val Thr

65 70 75 80

Phe Glu Leu Leu Phe Asp Gly Thr Asn Pro Ser Thr Glu Glu Met Gly

85 90 95

Asp Asp Phe Arg Ser Gly Leu Cys Pro Phe Asp Thr Ser Pro Val Val

100 105 110

Glu Gly Lys Tyr Asn Thr Thr Leu Leu Asn Gly Ser Ala Phe Tyr Leu

115 120 125

Val Cys Pro Ile Gly Trp Thr Gly Val Ile Glu Cys Thr Ala Val Ser

130 135 140

Pro Thr Thr Leu Arg Thr Glu Val Val Lys Thr Phe Arg Arg Asp Lys

145 150 155 160

Pro Phe Pro His Arg Met Asp Cys Val Thr Thr Thr Val Glu Asn Glu

165 170 175

Asp Leu Phe Tyr Cys Lys Leu Gly Gly Asn Trp Ala Cys Val Lys Gly

180 185 190

Glu Pro Ala Val Tyr Thr Arg Gly Val Val Glu Gln Arg Arg Trp Cys

195 200 205

Gly Phe Asp Phe Asp Gly Pro Asp Gly Leu Pro His Tyr Pro Ile Gly

210 215 220

Lys Cys Ile Leu Ala Asn Glu Thr Gly Tyr Arg Ile Val Asp Ser Thr

225 230 235 240

Asp Cys Asn Arg Asp Gly Val Val Ile Ser Thr Glu Gly Ser His Glu

245 250 255

Cys Leu Ile Gly Asn Thr Thr Val Lys Val His Ala Ser Asp Glu Arg

260 265 270

Leu Gly Pro Met Pro Cys Arg Pro Lys Glu Ile Val Ser Ser Ala Gly

275 280 285

Pro Val Met Lys Thr Ser Cys Thr Phe Asn Tyr Thr Lys Thr Leu Lys

290 295 300

Asn Arg Tyr Tyr Glu Pro Arg Asp Ser Tyr Phe Gln Gln Tyr Met Leu

305 310 315 320

Lys Gly Glu Tyr Gln Tyr Trp Phe Asp Leu Asp Ala Thr Asp His His

325 330 335

Ser Asp Tyr Phe Ala Glu Phe Asn Phe Asp Leu Leu Lys Leu Ala Gly

340 345 350

Asp Val Glu Ser Asn Pro Gly Pro Met Glu Asn Ile Thr Gln Trp Asn

355 360 365

Leu Ser Asp Asn Gly Thr Asn Gly Ile Gln His Ala Met Tyr Phe Arg

370 375 380

Gly Val Asn Arg Ser Leu His Gly Ile Trp Pro Gly Lys Ile Cys Lys

385 390 395 400

Gly Val Pro Thr His Leu Ala Thr Asp Val Glu Leu Lys Glu Ile Gln

405 410 415

Gly Met Met Asp Ala Ser Glu Gly Thr Asn Tyr Thr Cys Cys Lys Leu

420 425 430

Gln Arg His Glu Trp Asn Lys His Gly Trp Cys Asn Trp His Asn Ile

435 440 445

Asp Pro Trp Ile Gln Leu Met Asn Arg Thr Gln Ala Asp Leu Ala Glu

450 455 460

Gly Pro Pro Val Lys Glu Cys Ala Val Thr Cys Arg Tyr Asp Lys Asp

465 470 475 480

Ala Asp Ile Asn Val Val Thr Gln Ala Arg Asn Arg Pro Thr Thr Leu

485 490 495

Thr Gly Cys Lys Lys Gly Lys Asn Phe Ser Phe Ala Gly Thr Val Ile

500 505 510

Glu Ser Pro Cys Asn Phe Asn Val Ser Val Glu Asp Thr Leu Tyr Gly

515 520 525

Asp His Glu Cys Gly Ser Leu Leu Gln Asp Ala Ala

530 535 540

Claims (6)

1. silkworm with recombinant baculovirus Bmgp64-E2-2A-E0, this virus is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, and deposit number is CGMCC No.5604.

2. the preparation method of the said silkworm with recombinant baculovirus Bmgp64-E2-2A-E0 of claim 1 is characterized in that, said method comprising the steps of:

(a) method by pcr amplification obtains baculovirus envelope protein gp64 signal peptide gene sequence SP and baculovirus envelope protein gp64 membrane-spanning domain gene order TM, through BamH I/ EcoR I with XhoI/ HindThe III double digestion inserts pFastBacI carrier MCS upstream and downstream two ends, makes up surface display system transfer vector pFastBacI-gp64;

(b) adopting the pvL1393-E2-2A-E0 recombinant plasmid is template, is that primer carries out pcr amplification with SEQ ID:NO.6 and SEQ ID:NO.7, gene fusion construct E2-2A-E0;

(c) on fusion gene E2-2A-E0 double digestion product cloning to step (a) the gained surface display system transfer vector pFastBacI-gp64 with step (b) gained, and screening positive clone, obtain to shift recombinant plasmid pFastBacI-gp64-E2-2A-E0;

(d) step (c) gained is shifted recombinant plasmid pFastBacI-gp64-E2-2A-E0 transformed into escherichia coli DH10Bac competent cell; Carry out the screening of blue hickie on the LB culture plate of kantlex, qingfengmeisu qiong, tsiklomitsin, X-gal and IPTG containing; Picking hickie behind the lucifuge cultivation 40-48h, bacterium liquid dilution 10 behind the cultivation 16-20h ⁶Again coated plate is done screening, after picking hickie to LB substratum shakes bacterium cultivation 16-20h, with Virahol extracting recombinant baculovirus genome, and is PCR with the M13 universal primer and identifies;

(e) get step (d) the successful recombinant virus genomes of identifying through liposome mediated-method transfection silkworm BmN cell; The morbidity back obtains zero for viral suspension 0-4 ℃ preservation; Extract viral genome and carry out the PCR evaluation, obtain said silkworm with recombinant baculovirus Bmgp64-E2-2A-E0 with the M13 universal primer.

3. the said silkworm with recombinant baculovirus Bmgp64-E2-2A-E0 of a claim 1 fusion rotein of expressing, its aminoacid sequence is shown in SEQ ID:NO.11.

4. the said proteic preparation method of claim 3 is characterized in that, said method comprising the steps of:

(a) the described recombinant baculovirus Bmgp64-E2-2A-E0 of claim 3 infected silkworm BmN cell is carried out virus amplification;

(b) with stab inoculation inoculation silkworm five-age larva, pupa;

(c) collect the right 3 said albumen of expressing.

5. the application of the said silkworm with recombinant baculovirus Bmgp64-E2-2A-E0 of claim 1 in the swine Fever Vaccine preparation.

6. the application of the said albumen of claim 3 in the swine Fever Vaccine preparation.

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