CN106110319B - Preparation method of classical swine fever virus E2 gene recombinant baculovirus inactivated vaccine - Google Patents
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Abstract
The invention relates to a classical swine fever virus E2 gene recombinant baculovirus inactivated vaccine and a preparation method thereof. The preparation method of the classical swine fever virus E2 gene recombinant baculovirus inactivated vaccine comprises the following steps: 1) the recombinant baculovirus with the optimized sequence of the hog cholera virus E2 gene can effectively express the hog cholera virus E2 protein; 2) the prepared vaccine can be identified and detected after being immunized: the expressed E2 protein and the selected adjuvant are prepared into the inactivated vaccine, because the inactivated vaccine is not whole virus immunity, the identification and detection can be carried out, and the substance guarantee is provided for the purification of the swine fever in the future.
Description
Technical Field
The invention relates to a preparation method of a classical swine fever virus E2 gene recombinant baculovirus inactivated vaccine, belonging to the field of biological products for livestock.
Technical Field
Classical Swine Fever (CSF) is a highly lethal and contagious disease of Swine caused by Classical Swine Fever Virus (CSFV), and seriously harms the global Swine industry. China implements a control strategy mainly based on vaccination. The hog cholera lapinized virus live vaccine is safe to use, good in immune effect and stable in genetic character, and makes an important contribution to prevention and control of the hog cholera in China. However, the whole virus vaccine has the defects of the whole virus vaccine, and the whole virus vaccine cannot be identified with field wild virus infection after being immunized, thereby bringing difficulty to the purification of the swine fever. The classical swine fever virus subunit vaccine can be used for differential diagnosis with field wild virus infection due to single immune antigen component, and has very important significance for epidemic situation determination.
A genetic engineering Subunit vaccine (Subunit vaccine) is prepared by connecting and recombining a genome for coding a certain specific protein with a proper vector, introducing the recombined genome into a receptor cell (prokaryotic or eukaryotic cell), efficiently expressing the recombined genome in a host cell, and adding a proper adjuvant. In swine feverThe E2 protein in the virus is a main protective antigen, can induce the organism to generate a neutralizing antibody and protect the organism from being attacked by strong virus, so the E2 gene is the first choice target gene in the research of genetic engineering vaccines. Hulst et al (Hulst et al, 1993) insert the envelope protein gene of classical swine fever virus E2 (then referred to as E1 in the literature) into a baculovirus vector, which is then expressed in insect cells, and immunize swine with the expression product against 100LD50The attack of the virulent strain of the classical swine fever virus Brescia invents a novel and safe classical swine fever subunit vaccine. Bouma et al (Bouma et al, 2000) constructed the subunit vaccine of classical swine fever virus E2, and 10 days after immunizing test pigs, it can produce good antibody level, can resist the attack of virulent strain, can be used as the selection object of emergency immunization after the outbreak of classical swine fever. Moormann et al (Moormann et al, 2000) also used E2 protein as a research object, and added with a second envelope protein Erns to construct an E2 subunit marker vaccine, which can resist 100LD after 2 weeks of animal immunization50The attack of strong toxicity does not appear clinical symptoms, and the strong toxicity can be prevented within 3 weeks to 6 months after immunization; the Erns antibody can be detected 14d after immunization, and vaccine immunized pigs and wild virus infected pigs can be distinguished.
At present, few commercial genetic engineering subunit vaccines are available. Bayer corporation developed the first classical swine fever virus E2 gene engineering recombinant subunit vaccineCSF is matched with ELISA detection kit capable of distinguishing immunity from natural infection, and said vaccine can produce neutralizing antibody and resist virulent attack 14 days after pig is immunized. Classical swine fever E2 gene recombinant subunit vaccine developed by Ziegler and other baculovirus as expression vectorPesti (Ziegler et al, 2002) was also successful, and secondary immunizations at 4 weeks after the first immunizations resulted in robust immunity, as well as a matched ELISA test kit to distinguish between immunization and natural infection. The domestic Xinjiang Tiankang animal biotechnology GmbH also develops the swine fever E2 gene recombinant subunit vaccine (CN103908663A), but still has room for improvement.
The related documents are:
Hulst M.M.,Westra D.F.,Wensvoort G.,Moormann R.J.,1993,Glycoprotein E1 of hog cholera virus expressed in insect cells protects swine from hogcholera.J Virol 67,5435-5442.
Bouma A.,De Smit A.J.,De Jong M.C.,De Kluijver E.P.,Moormann R.J.,2000,Determination of the onset of the herd-immunity induced by the E2sub-unit vaccine against classical swine fever virus.Vaccine 18,1374-1381
Moormann R.J.,Bouma A.,Kramps J.A.,Terpstra C.,De Smit,H.J.,2000,Development of a classical swine fever subunit marker vaccine and companiondiagnostic test.Vet Microbiol 73,209-219.
Ziegler U.,Kaden V.,2002,Vaccination of weaner pigs against classical swine fever with the subunit vaccine"Porcilis Pesti":influence of differentimmunization plans on excretion and transmission of challenge virus.BerlMunch Tierarztl Wochenschr 115,267-273.
xinjiang Tiankang animal biotechnology, Inc., a novel classical swine fever virus E2 recombinant baculovirus inactivated vaccine and a preparation method thereof, CN103908663A
Disclosure of Invention
The invention aims to optimize the sequence of hog cholera virus E2 gene by adopting a biotechnology means and construct recombinant baculovirus for double expression so as to obtain soluble E2 protein, and the subunit vaccine is prepared by inactivation treatment, addition of a proper adjuvant and emulsification. Provides convenience for differential diagnosis of the immunized and the field epidemic virus infection.
The technical scheme of the invention is as follows:
1. the preparation method of the inactivated vaccine of the swine fever virus E2 gene recombinant baculovirus is characterized in that the vaccine is prepared by taking a constructed baculovirus BacVDMOSE2 strain as a production strain, the strain is proposed to be classified and named as alfalfa silver mosquito noctuid nuclear polyhedrosis virus, the strain is delivered to China Committee for microorganism preservation, China Committee for microorganism Collection, Ministry of microbiology, North City, Tokyo, West Lu No.1 Hokko 3, Naja, Beijing, on 2016 (06 months 01), and the preservation numbers are respectively: CGMCC No. 12546.
2. The method for preparing the hog cholera virus E2 gene recombinant baculovirus inactivated vaccine as claimed in claim 1, wherein the constructed baculovirus BacVDMOSE2 strain carries Mels-optiSE2-1 (SEQ ID NO: 1) and Mels-optiSE2-2 (SEQ ID NO: 2).
3. The process for preparing the inactivated vaccine of the classical swine fever virus E2 gene recombinant baculovirus as claimed in claim 1, wherein the baculovirus strain BacVDMOSE2 constructed by the method is used for expression to obtain soluble E2 protein, and the subunit vaccine is prepared after inactivation and emulsification by adding a proper adjuvant.
Main technical principle of the invention
The optimized E2 gene sequence is fused with a secretion signal peptide, cloned into a baculovirus genome through a transposition technology to construct a recombinant baculovirus, and E2 protein is obtained, added with a proper adjuvant and a protective agent, and emulsified to prepare the subunit vaccine.
Detailed description of the invention
1. Synthesis of optimized sequence of hog cholera virus E2 gene
The gene sequence of the hog cholera virus Shimen Strain (SM) E2 is optimized, an SM strain E2 gene optimized nucleotide sequence is obtained by an artificial synthesis technology and is cloned to a pMD 18 vector, and the name is as follows: pMD-SE 2.
2. Fusion of hog cholera virus E2 gene and signal peptide sequence
Extracting pMD-SE2 plasmid, adding proper restriction enzyme cutting sites through primers, respectively amplifying SE2 and signal peptide Mels gene through PCR technology, obtaining fusion gene with signal peptide Mels gene through overlap extension PCR technology (overlap extension PCR technology and application thereof in genetic engineering. molecular plant breeding, 2006,05: 747-. The constructed fusion gene plasmids are respectively named as: pMOSE2-1, pMOSE 2-2. The method comprises the following specific steps:
optimized SM strain fusion gene 1: Mels-optiSE2-1 (abbreviated as MOSE2-1) (sequence 1)
BamHI site is added at the upstream, and stop codon and HindIII site are added at the downstream
Optimized SM strain fusion gene 2 Mels-optiSE2-2 (abbreviated as MOSE2-2) (sequence 2)
SmaI restriction site is added at the upstream, and a stop codon and KpnI site are added at the downstream
4. Subcloning into baculovirus transfer vectors
pMOSE2-1 and pFastBacDual are respectively subjected to double digestion, purified and recovered, the digestion fragment MOSE2-1 is connected with pFastBacDual linear plasmid, transformed and identified, and the obtained new plasmid is named as pSMOSE2(pS represents a single expression plasmid). Then, pMOSE2-2 and pSMOSE2 were subjected to double digestion, purified and recovered, the digested fragment MOSE2-2 was ligated with pSMOSE2 linear plasmid, transformed, identified, and the new plasmid obtained was named pDMOSE2(pD represents a double expression plasmid).
5. Transposition to obtain recombinant bacmid DNA
The bacmid DNA constructed by DH10Bac transformed with pSMOSE2 and pDMOSE2 was named: bSMOSE2, bDMOSE 2.
6. Obtaining of recombinant viruses
Recombinant bacmid bSMOSE2 and bDMOSE2 transfect sf9 cells by using transfection reagent Cellffectin respectively, enlarge culture, collect culture supernatant and carry out PCR identification. Two strains of recombinant viruses BacVSMOSE2 strain and BacVDMOSE2 strain are obtained, the suggested classification names of the two strains are alfalfa silver mosquito noctuid nuclear polyhedrosis virus, wherein the BacVDMOSE2 strain is delivered to China general microbiological culture collection center of microbiological culture Collection institute of China academy of sciences, North Asian province No.1 Hokko 3 of the sunward region, Beijing city, on 2016, 06 and 01 days, and the preservation number is CGMCC No. 12546.
7. Expression analysis
And (5) carrying out subculture expansion on the identified viruses, wherein the second generation is marked as P2Stock, and the third generation is marked as P3 Stock. The first three generations were used as seed virus, and the culture supernatant of the 4 th 96h generation was used for ELISA analysis (see FIG. 1). The result shows that the recombinant viruses can secrete E2 protein, but the expression level of the baculovirus BacVDMOSE2 strain (double expression) is obviously higher than that of the BacVSMOSE2 strain (single expression).
8. Emulsifying and preparing seedling
Taking the classical swine fever virus E2 recombinant baculovirus BacVDMOSE2 strain expression protein vaccine as an example. The correctly identified seed virus was expanded to P6 passages using sf9 cells and virus titers were determined. Performing suspension culture on the High Five insect cells by using an ExpressFive culture medium (shaking culture at 27 ℃ and 180 r/min), replacing a new ExpressFive culture medium when the High Five insect cells grow to a logarithmic growth phase, and adjusting the cell concentration to be 2-5 multiplied by 10-6The recombinant baculovirus BacVDMOSE2 was infected with cells at 1MOI dose and harvested 96 hours later. Centrifuging at 2000r/min for 10min, collecting supernatant, performing SDS-PAGE electrophoresis, and calculating the content of the target product. Adding BEI into antigen with final concentration of lmmol/L, and inactivating at 37 deg.C for 48h to inactivate recombinant virus. Mixing the expression supernatant with an ISA206 adjuvant at a ratio of 1: l, and homogenizing the antigen liquid adjuvant by an emulsifying machine at 12000r/min for 10min to obtain the classical swine fever virus E2 recombinant baculovirus inactivated vaccine.
9. Hog cholera virus E2 gene recombination baculovirus inactivated vaccine swine body immunity test
Taking 20 healthy pigs with swine fever antibodies as negative, wherein 12 healthy pigs immunize 3 batches of swine fever virus E2 gene recombinant baculovirus inactivated vaccines, 4 immune swine fever lapinized attenuated viruses (subculture cell sources), and the other 4 healthy pigs are control groups, the neck of the back of each ear of the immune group is injected with swine fever virus E2 gene recombinant baculovirus inactivated vaccine 2m1, the second immunization is carried out 21d after the first immunization in the same dose and mode, blood is collected once every 7 days during the first immunization and the second immunization, and serum is separated to measure the antibody level. The results demonstrate that the inactivated vaccine has substantially the same level of antibody as the live vaccine, demonstrating comparable immunopotency. (see FIG. 2)
10. Safety test of classical swine fever virus E2 gene recombinant baculovirus inactivated vaccine
After 2 times of dose (4m 1/head) of the hog cholera virus E2 gene recombinant baculovirus inactivated vaccine is used for immunizing a pig, no local and systemic adverse reaction is seen.
Information on microbial resources related to the present invention
Hog cholera virus Shimada virulent strain (SM strain, reviewed in the institute of veterinary medicine, China center for veterinary culture Collection of microorganisms, China catalog of veterinary cultures (second edition), China Press for agricultural science and technology, 2002, p 145); the recombinant virus name is: BacVDMOSE2, wherein the male parent is baculovirus genome from DH10Bac, the fusion gene of CSFV SM strain E2 gene and secretory signal peptide Mels is cloned into two reading frames of pFastBacDual plasmid in full sequence, and recombined with baculovirus genome in DH10Bac to obtain new baculovirus BacVDMOSE2, which is named as alfalfa silver mosquito noctuid nuclear polyhedrosis virus, and the virus is delivered to 2016, Sai, China Committee for microbiological Collection of microorganisms, 2016, No. 4, Sai, Ten, Beijing, to 2016, Sai, Japan institute of microbiology, China academy for microbiological research, China, having accession number of CGMCC No.12546, on 01 month in 06 years.
Drawings
FIG. 1 analysis of the expression of classical swine fever virus E2 recombinant baculovirus, which proves that 4 recombinant viruses can express fusion gene.
FIG. 2 shows the growth curve of the immune antibody of the inactivated vaccine against classical swine fever virus E2 gene recombinant baculovirus
FIG. 3 is a construction scheme of a recombinant baculovirus having the sequence of classical swine fever virus E2 gene shown in the technical scheme of the present invention
THE ADVANTAGES OF THE PRESENT INVENTION
The invention relates to a classical swine fever virus E2 gene recombinant baculovirus inactivated vaccine and a preparation method thereof. The preparation method of the classical swine fever virus E2 gene recombinant baculovirus inactivated vaccine comprises the following steps: 1) the recombinant baculovirus with the optimized sequence of the hog cholera virus E2 gene can effectively express the hog cholera virus E2 protein; 2) the prepared vaccine can be identified and detected after being immunized: the expressed E2 protein and the selected adjuvant are prepared into the inactivated vaccine, because the inactivated vaccine is not whole virus immunity, the identification and detection can be carried out, and the substance guarantee is provided for the purification of the swine fever in the future.
Examples
Example 1 construction of recombinant Virus baculoviruses BacVSMOSE2 and BacVDMOSE2 strains
1. Synthesis of optimized sequence of hog cholera virus E2 gene
The gene sequence of the hog cholera virus Shimen Strain (SM) E2 is optimized, an SM strain E2 gene optimized nucleotide sequence is obtained by an artificial synthesis technology and is cloned to a pMD 18 vector, and the name is as follows: pMD-SE 2.
2. Fusion of hog cholera virus E2 gene and signal peptide sequence
Extracting pMD-SE2 plasmid, adding proper restriction enzyme cutting sites through primers, respectively amplifying SE2 and signal peptide Mels gene through PCR technology, obtaining fusion gene with signal peptide Mels gene through overlap extension PCR technology (overlap extension PCR technology and application thereof in genetic engineering. molecular plant breeding, 2006,05: 747-. The constructed fusion gene plasmids are respectively named as: pMOSE2-1, pMOSE 2-2. The method comprises the following specific steps:
optimized SM strain fusion gene 1: Mels-optiSE2-1 (abbreviated as MOSE2-1) (sequence 1)
BamHI site is added at the upstream, and stop codon and HindIII site are added at the downstream
Optimized SM strain fusion gene 2 Mels-optiSE2-2 (abbreviated as MOSE2-2) (sequence 2)
SmaI restriction site is added at the upstream, and a stop codon and KpnI site are added at the downstream
4. Subcloning into baculovirus transfer vectors
pMOSE2-1 and pFastBacDual are respectively subjected to double digestion, purified and recovered, the digestion fragment MOSE2-1 is connected with pFastBacDual linear plasmid, transformed and identified, and the obtained new plasmid is named as pSMOSE2(pS represents a single expression plasmid). Then, pMOSE2-2 and pSMOSE2 were subjected to double digestion, purified and recovered, the digested fragment MOSE2-2 was ligated with pSMOSE2 linear plasmid, transformed, identified, and the new plasmid obtained was named pDMOSE2(pD represents a double expression plasmid).
5. Transposition to obtain recombinant bacmid DNA
The bacmid DNA constructed by DH10Bac transformed with pSMOSE2 and pDMOSE2 was named: bSMOSE2, bDMOSE 2.
6. Obtaining of recombinant viruses
Recombinant bacmid bSMOSE2 and bDMOSE2 transfect sf9 cells by using transfection reagent Cellffectin respectively, enlarge culture, collect culture supernatant and carry out PCR identification. Two strains of recombinant viruses BacVSMOSE2 strain and BacVDMOSE2 strain are obtained, the suggested classification names of the two strains are alfalfa silver mosquito noctuid nuclear polyhedrosis virus, wherein the BacVDMOSE2 strain is delivered to China general microbiological culture collection center of microbiological culture Collection institute of China academy of sciences, North Asian province No.1 Hokko 3 of the sunward region, Beijing city, on 2016, 06 and 01 days, and the preservation number is CGMCC No. 12546.
7. Expression analysis
And (5) carrying out subculture expansion on the identified viruses, wherein the second generation is marked as P2Stock, and the third generation is marked as P3 Stock. The first three generations were used as seed virus, and the culture supernatant of the 4 th 96h generation was used for ELISA analysis (see FIG. 1). The result shows that the recombinant viruses can secrete E2 protein, but the expression level of the baculovirus BacVDMOSE2 strain (double expression) is obviously higher than that of the BacVSMOSE2 strain (single expression).
Example 2-preparation of classical swine fever virus E2 recombinant baculovirus inactivated vaccine
Amplifying the correctly identified classical swine fever virus E2 recombinant baculovirus BacVDMOSE2 strain to P6 generation by sf9 cell, and using TCID50The method determines the virus titer. Culturing High Five insect cells (Invitrogen) in suspension in ExpressFive culture medium (Invitrogen), culturing at 27 deg.C under shaking at 180r/min, changing new ExpressFive culture medium when the High Five insect cells grow to logarithmic phase, adjusting cell concentration to 2-5 × 106The recombinant baculovirus BacVDMOSE2 strain was infected with cells at 1MOI dose and harvested 96 hours later. Centrifuging at 2000r/min for 10min, and collectingAnd (5) carrying out SDS-PAGE electrophoresis on the supernatant, and calculating the content of the target product. Adding an inactivating agent BEI into the antigen with the final concentration of lmmol/L, and inactivating the antigen at 37 ℃ for 48h to inactivate the recombinant virus. Mixing the expression supernatant with an ISA206 adjuvant at a ratio of 1: l, and homogenizing the antigen liquid adjuvant by an emulsifying machine at 12000r/min for 10min to obtain the classical swine fever virus E2 recombinant baculovirus inactivated vaccine.
Example 3-hog cholera virus E2 gene recombinant baculovirus inactivated vaccine swine body immunoassay
Taking 20 healthy pigs with swine fever antibodies as negative, wherein 12 healthy pigs immunize 3 batches of swine fever virus E2 recombinant baculovirus inactivated vaccines, 4 immune swine fever lapinized attenuated viruses (subculture cell sources), and the other 4 healthy pigs are control groups, the neck of the back of each ear of the immune group is injected with swine fever virus E2 recombinant baculovirus inactivated vaccine 2m1, the second immunization is carried out 21d after the first immunization in the same dose and mode, blood is collected once every 7 days during the first immunization and the second immunization, and serum is separated to measure the antibody level. The results demonstrate that the inactivated vaccine has substantially the same level of antibody as the live vaccine, demonstrating comparable immunopotency. (see FIG. 2)
Example 4
Safety test of inactivated vaccine of recombinant baculovirus of classical swine fever virus E2 gene
After 2 times of dose (4m 1/head) of the hog cholera virus E2 recombinant baculovirus inactivated vaccine is used for immunizing pigs, local and systemic adverse reactions such as fever, diarrhea, anorexia, local injection swelling and the like are not caused.
Example 5
Construction of recombinant baculovirus BacVSMOCE2 Strain and BacVDMOCE2 Strain
According to the same principle of constructing the classical swine fever virus E2 recombinant baculovirus BacVDMOSE2 strain, the invention simultaneously constructs the recombinant baculovirus containing the classical swine fever lapinized virus (C strain) E2 gene.
1. Synthesis of optimized sequence of hog cholera lapinized virus (C strain) E2 gene
The E2 gene sequence of hog cholera lapinized virus (C strain) is optimized, the optimized nucleotide sequence of the C strain E2 gene is obtained by a synthetic technology and is cloned to a pMD 18 vector, and the name is as follows: pMD-CE 2.
2. Fusion of hog cholera attenuated E2 gene and signal peptide sequence
Extracting pMD-CE2 plasmid, adding proper restriction enzyme cutting sites through primers, amplifying CE2 and signal peptide Mels gene through PCR technology, obtaining fusion gene with the signal peptide Mels gene through overlap extension PCR technology, and cloning to pMD 18 vector. The constructed fusion gene plasmids are respectively named as: pMOCE2-1, pMOCE 2-2. The method comprises the following specific steps:
optimized C strain fusion gene 1: Mels-optiCE2-1 (abbreviated as MOCE2-1) (sequence 3)
BamHI site is added at the upstream, and stop codon and HindIII site are added at the downstream
Optimized C strain fusion gene 2 Mels-optiCE2-2 (abbreviated as MOCE2-2) (sequence 4)
SmaI restriction site is added at the upstream, and a stop codon and KpnI site are added at the downstream
4. Subcloning into baculovirus transfer vectors
pMOCE2-1 and pFastBacDual are respectively subjected to double digestion, purified and recovered, the digestion fragment MOCE2-1 is connected with pFastBacDual linear plasmid, transformed and identified, and the obtained new plasmid is named pSMOCE2(pS represents single expression plasmid). Then, pMOCE2-2 and pSMOCE2 were digested simultaneously, purified and recovered, the digested fragment MOCE2-2 was ligated with pSMOCE2 linear plasmid, transformed and identified, and the new plasmid obtained was named pDMOCE2(pD represents the double expression plasmid).
5. Transposition to obtain recombinant bacmid DNA
The bacmid DNA constructed by DH10Bac transformed with pSMOCE2 and pDMOCE2 was named: bSMOCE2, bDMOCE 2.
6. Obtaining of recombinant viruses
Recombinant bacmid bSMOCE2 and bDMOCE2 transfect sf9 cells by using transfection reagent Cellffectin respectively, and the recombinant bacmid bSMOCE2 and the recombinant bacmid bDMOCE2 are subjected to amplification culture, culture supernatants are collected and subjected to PCR identification. Two strains of recombinant viruses are obtained and named as baculovirus BacVSMOCE2 strain and BacVDMOCE2 strain respectively, and the suggested classification names are the autographa californica nuclear polyhedrosis virus.
7. Expression analysis
And (5) carrying out subculture expansion on the identified viruses, wherein the second generation is marked as P2Stock, and the third generation is marked as P3 Stock. The first three generations were used as seed virus, and the culture supernatant of the 4 th 96h generation was used for ELISA analysis (see FIG. 1). The result shows that the recombinant virus BacVSMOCE2 strain and BacVDMOCE2 strain can secrete E2 protein and can be used for producing classical swine fever virus E2 recombinant baculovirus inactivated vaccines, but the expression quantity of the BacVSMOCE2 strain (double expression) is obviously higher than that of the BacVSMOCE2 strain (single expression).
Claims (3)
1. The preparation method of the inactivated vaccine of the swine fever virus E2 gene recombinant baculovirus is characterized in that the vaccine is prepared by taking a constructed baculovirus BacVDMOSE2 strain as a production strain, the strain is proposed to be classified and named as alfalfa silver mosquito noctuid nuclear polyhedrosis virus, the strain is delivered to China Committee for culture Collection of microorganisms of Ministry of microbiology of Ministry of China, Ministry of microbiology, Naja, No.1 Hokko, Beijing, No. 3, in 2016 (06, 01), and the preservation numbers are as follows: CGMCC No. 12546.
2. The method for preparing the hog cholera virus E2 gene recombinant baculovirus inactivated vaccine as claimed in claim 1, wherein the baculovirus strain BacVDMOSE2 is constructed to carry Mels-optiSE2-1 as shown in sequence 1 and Mels-optiSE2-2 as shown in sequence 2.
3. The process for preparing the inactivated vaccine of the classical swine fever virus E2 gene recombinant baculovirus as claimed in claim 1, wherein the baculovirus strain BacVDMOSE2 constructed by the method is used for expression to obtain soluble E2 protein, and the subunit vaccine is prepared after inactivation and emulsification by adding a proper adjuvant.
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