CN106110319B - Preparation method of classical swine fever virus E2 gene recombinant baculovirus inactivated vaccine - Google Patents

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

Preparation method of swine fever virus E2 gene recombinant baculovirus inactivated vaccine

technical field

The invention relates to a method for preparing a recombinant baculovirus inactivated vaccine of classical swine fever virus E2 gene, which belongs to the field of veterinary biological products.

technical background

Classical Swine Fever (CSF) is a highly lethal and contagious infectious disease of pigs caused by Classical swine fever virus (CSFV), which seriously endangers the global pig industry. my country implements a control strategy based on vaccination. The attenuated live vaccine of swine fever rabbit is safe to use, has good immune effect and stable genetic traits, and has made important contributions to the prevention and control of swine fever in my country. However, the whole virus vaccine also has its own weakness. After immunization, it cannot be distinguished from field virus infection, which brings difficulties for the purification of swine fever. The subunit vaccine of classical swine fever virus has a single immune antigen component, so it can be differentially diagnosed from wild virus infection in the field, which is of great significance for the determination of the epidemic situation.

Genetic engineering subunit vaccine (Subunit vaccine) is to connect and recombine the genome encoding a specific protein with an appropriate vector, and then introduce it into recipient cells (prokaryotic or eukaryotic cells) to make it highly expressed in the host cell, and then add suitable adjuvant for the vaccine. In classical swine fever virus, E2 protein is the main protective antigen, which can induce the body to produce neutralizing antibodies and protect the body from virulent attacks. Therefore, E2 gene is the preferred target gene in the research of genetic engineering vaccines. Hulst et al. (Hulst et al., 1993) inserted the envelope protein gene of classical swine fever virus E2 (referred to as E1 in the literature at the time) into a baculovirus vector, and then expressed it in insect cells. The expression product immune pigs could resist 100LD ₅₀ swine fever virus Brescia In response to the attack of the virulent strain, a new type and safe subunit vaccine of classical swine fever was invented. Bouma et al. (Bouma et al., 2000) constructed the E2 subunit vaccine of classical swine fever virus, which can produce a good antibody level 10 days after the immunization test pigs, can resist the attack of strong virus strains, and can be used as an emergency immunization vaccine after the outbreak of classical swine fever. Select an object. Moormann et al. (Moormann et al., 2000) also used the E2 protein as the research object, and at the same time added the second envelope protein Erns to construct the E2 subunit-labeled vaccine. Two weeks after the animal was immunized, it could produce a 100LD ₅₀ -dose virulent No clinical symptoms appear, and they can be protected from the strong virus attack within 3 weeks to 6 months after immunization; Erns antibodies can be detected 14 days after immunization, which can distinguish vaccine-immunized pigs from wild virus-infected pigs.

At present, there are not many genetically engineered subunit vaccines commercially available. Bayer developed the first genetically engineered recombinant subunit vaccine against classical swine fever virus E2 CSF is equipped with an ELISA detection kit that can distinguish immunity from natural infection. The vaccine can produce neutralizing antibodies and resist virulent challenge 14 days after immunization of pigs. The swine fever E2 gene recombinant subunit vaccine developed by Ziegler et al. using baculovirus as an expression vector Pesti (Ziegler et al., 2002) is also relatively successful. The second immunization at an interval of 4 weeks after the first immunization can produce strong immunity. There is also a matching ELISA detection kit for distinguishing immunity from natural infection. Domestic Xinjiang Tiankang Animal Husbandry Biotechnology Co., Ltd. has also developed the recombinant subunit vaccine (CN103908663A) of the swine fever E2 gene, but there is still room for improvement.

Related literature:

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 companion diagnostic 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 different immunization plans on excretion and transmission of challenge virus. Berl Munch Tierarztl Wochenschr 115, 267-267

Xinjiang Tiankang Animal Husbandry Biotechnology Co., Ltd., a novel swine fever virus E2 recombinant baculovirus inactivated vaccine and its preparation method, CN103908663A

Contents of the invention

The purpose of the present invention is to use biotechnological means to optimize the sequence of the E2 gene of classical swine fever virus and construct a recombinant baculovirus for dual expression to obtain soluble E2 protein, which is inactivated and added with a suitable adjuvant, and then emulsified to prepare subunits vaccine. It is convenient for the differential diagnosis between immunization and field epidemic virus infection.

Technical scheme of the present invention is:

1. The preparation method of the recombinant baculovirus inactivated vaccine of classical swine fever virus E2 gene is characterized in that the vaccine is prepared from the constructed baculovirus BacVDMOSE2 strain as a production strain, and the proposed classification of the strain is named as silver mosquito Noctuid moth nuclear polyhedrosis virus, the strain was sent to the Ordinary Microorganism Collection Center of China Microbiology Collection Committee, Institute of Microbiology, Chinese Academy of Sciences, on June 1, 2016, No. 1, Beichen West Road, Chaoyang District, Beijing. For: CGMCCNo.12546.

2. the described swine fever virus E2 gene recombinant baculovirus inactivated vaccine preparation method of claim 1 is characterized in that carrying Mels-optiSE2-1 (sequence 1) and Mels-optiSE2-2 in the baculovirus BacVDMOSE2 strain of its construction (sequence 2).

3. the preparation method of swine fever virus E2 gene recombinant baculovirus inactivated vaccine as claimed in claim 1 is characterized in that the baculovirus BacVDMOSE2 strain of its construction is used to express and obtain soluble E2 protein, through inactivation, add suitable adjuvant The subunit vaccine was prepared after emulsification.

Main technical principle of the present invention

Refer to the E2 gene sequence of classical swine fever virus, optimize and synthesize it, fuse the optimized E2 gene sequence with the secretion signal peptide, and clone it into the baculovirus genome through transposition technology, construct a recombinant baculovirus, obtain E2 protein, and add a suitable adjuvant And protective agent, prepared into subunit vaccine after emulsification.

Specific embodiments of the invention

1. Synthesis of optimized sequence of classical swine fever virus E2 gene

The E2 gene sequence of classical swine fever virus Shimen strain (SM) was optimized, and the optimized nucleotide sequence of SM strain E2 gene was obtained by artificial synthesis technology, cloned into the pMD 18 vector, and named: pMD-SE2.

2. Fusion of classical swine fever virus E2 gene and signal peptide sequence

Extract the pMD-SE2 plasmid, add suitable restriction sites through primers, amplify SE2 and the signal peptide Mels gene by PCR technology, and then use overlap extension PCR technology (overlap extension PCR technology and its application in genetic engineering. Molecular Plant Breeding, 2006, 05:747-750) obtained the fusion gene with the signal peptide Mels gene, and cloned it into the pMD 18 vector. The constructed fusion gene plasmids were respectively named: pMOSE2-1, pMOSE2-2. details as follows:

Optimized SM strain fusion gene 1: Mels-optiSE2-1 (abbreviated as MOSE2-1) (SEQ ID NO: 1)

A BamHI restriction site is added upstream, and a stop codon and HindIII site are added downstream

Optimized SM strain fusion gene 2: Mels-optiSE2-2 (abbreviated as MOSE2-2) (sequence 2)

Add SmaI restriction site upstream, add stop codon and KpnI site downstream

4. Subcloning into Baculovirus Transfer Vectors

The pMOSE2-1 and pFastBacDual were subjected to double enzyme digestion, purified and recovered, the digested MOSE2-1 was connected with the pFastBacDual linear plasmid, transformed and identified, and the obtained new plasmid was named pSMOSE2 (pS indicates a single expression plasmid). Then, pMOSE2-2 and pSMOSE2 were subjected to double enzyme digestion respectively, purified and recovered, the enzyme digestion fragmented MOSE2-2 was connected with the pSMOSE2 linear plasmid, transformed, identified, and the obtained new plasmid was named pDMOSE2 (pD indicates double expression plasmid).

5. Transposition to obtain recombinant bacmid DNA

The bacmid DNA constructed by transforming pSMOSE2 and pDMOSE2 into DH10Bac respectively was named as: bSMOSE2 and bDMOSE2.

6. Acquisition of Recombinant Viruses

Recombinant bacmids bSMOSE2 and bDMOSE2 were transfected into sf9 cells with the transfection reagent Cellfectin respectively, expanded and cultured, and the culture supernatant was collected for PCR identification. Obtained two strains of recombinant virus baculovirus BacVSMOSE2 strain and BacVDMOSE2 strain, the suggested taxonomic names of the two strains are both Spodoptera californica nuclear polyhedrosis virus, and the baculovirus BacVDMOSE2 strain was sent to the hospital on June 01, 2016 Handed over to the General Microorganisms Collection Center of the Chinese Academy of Sciences, No. 1, Beichen West Road, Chaoyang District, Beijing, No. 3, Institute of Microbiology, Chinese Academy of Sciences, and China Microorganisms Collection Center, the preservation number is CGMCC No.12546.

7. Expression Analysis

The identified virus was subcultured and expanded, and the second generation was recorded as P2Stock, and the third generation was recorded as P3Stock. The first three generations were used as seed virus, and the 96h culture supernatant of the fourth generation was taken to coat the plates for ELISA analysis (see Figure 1). The results showed that all recombinant viruses could secrete E2 protein, but the expression level of baculovirus BacVDMOSE2 strain (double expression) was significantly higher than that of BacVSMOSE2 strain (single expression).

8. Emulsified seedlings

Take the recombinant baculovirus BacVDMOSE2 strain of classical swine fever virus E2 as an example. The correctly identified sf9 cells were expanded to the P6 generation, and the virus titer was determined. Use ExpressFive medium to suspend High Five insect cells (27°C, 180r/min shaking culture). When the High Five insect cells grow to the logarithmic growth phase, replace the new ExpressFive medium and adjust the cell concentration to 2～5×10 ^-6 /ml, the cells were infected with the recombinant baculovirus BacVDMOSE2 at a dose of 1 MOI, and harvested after 96 hours. Centrifuge at 2000r/min for 10min, take the supernatant, conduct SDS-PAGE electrophoresis, and calculate the content of the target product. The recombinant virus can be inactivated by adding BEI to the antigen at a final concentration of 1 mmol/L and inactivating it at 37°C for 48 hours. The expression supernatant was mixed with ISA206 adjuvant at a ratio of 1:1, and the antigen solution adjuvant was homogenized with an emulsifier at 12000 r/min for 10 minutes to prepare a recombinant baculovirus inactivated vaccine of classical swine fever virus E2.

9. Immunization test of swine fever virus E2 gene recombinant baculovirus inactivated vaccine in pigs

Take 20 healthy pigs negative for classical swine fever antibody, 12 of which were immunized with 3 batches of recombinant baculovirus inactivated vaccine of classical swine fever virus E2 gene, 4 were immunized with attenuated swine fever rabbit virus (passage cell source), and the other 4 were controls In the immunization group, each head of the immunization group was intramuscularly injected with 2 ml of the recombinant baculovirus inactivated vaccine of CSFV E2 gene, 21 days after the first immunization, the second immunization was carried out with the same dose and method, and blood was collected every 7 days during the first immunization and the second immunization. Once, the serum was separated to measure the antibody level. The results proved that the growth of the inactivated vaccine was basically the same as that of the live vaccine antibody level, which proved that the immune efficacy was equivalent. (See Figure 2)

10. Safety test of recombinant baculovirus inactivated vaccine of classical swine fever virus E2 gene

No local and systemic adverse reactions were observed after immunizing pigs with 2 times the dose (4ml/head) of the recombinant baculovirus inactivated vaccine of classical swine fever virus E2 gene.

Microorganism resource information involved in the present invention

Severe virulent strain of classical swine fever virus Shimen (SM strain, see China Veterinary Drug Control Institute, China Veterinary Microbiological Strain Preservation Management Center edited, Chinese Veterinary Strain Catalog (Second Edition), China Agricultural Science and Technology Press, 2002 , p145); the name of the recombinant virus is: BacVDMOSE2, and the male parent is the baculovirus genome from the DH10Bac bacteria, by cloning the full sequence of the fusion gene of the classical swine fever virus SM strain E2 gene and the secretion signal peptide Mels into the pFastBacDual plasmid for two reads After the code frame, the new baculovirus BacVDMOSE2 strain obtained after recombination with the baculovirus genome in the DH10Bac bacteria, the proposed classification is named as the californica nuclear polyhedrosis virus. On 01, it was sent to the General Microbiology Collection Center of China Microbiology Collection Committee, Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Courtyard, Beichen West Road, Chaoyang District, Beijing, with the preservation number CGMCC No.12546.

Description of drawings

Figure 1 Analysis of the expression of CSFV E2 recombinant baculovirus, which proves that all four recombinant viruses can express fusion genes.

Fig. 2 Growth curve of immune antibody of classical swine fever virus E2 gene recombinant baculovirus inactivated vaccine

Fig. 3 shows the construction route of the recombinant baculovirus of classical swine fever virus E2 gene sequence in the technical roadmap of the present invention

Advantages of the invention

The invention relates to a recombinant baculovirus inactivated vaccine of classical swine fever virus E2 gene and a preparation method thereof. In the preparation method of the swine fever virus E2 gene recombinant baculovirus inactivated vaccine of the present invention: 1) the recombinant baculovirus with the optimized sequence of the swine fever virus E2 gene can effectively express the swine fever virus E2 protein; 2) after the prepared vaccine is immunized Differential testing is possible: the expressed E2 protein and the selected adjuvant are used to prepare an inactivated vaccine. Because it is not immune to the whole virus, it can be used for differential testing to provide material guarantee for the purification of swine fever in the future.

Example

Embodiment 1 - the construction of recombinant virus baculovirus BacVSMOSE2 strain and BacVDMOSE2 strain

1. Synthesis of optimized sequence of classical swine fever virus E2 gene

The E2 gene sequence of classical swine fever virus Shimen strain (SM) was optimized, and the optimized nucleotide sequence of SM strain E2 gene was obtained by artificial synthesis technology, cloned into the pMD 18 vector, and named: pMD-SE2.

2. Fusion of classical swine fever virus E2 gene and signal peptide sequence

Extract the pMD-SE2 plasmid, add suitable restriction sites through primers, amplify SE2 and the signal peptide Mels gene by PCR technology, and then use overlap extension PCR technology (overlap extension PCR technology and its application in genetic engineering. Molecular Plant Breeding, 2006, 05:747-750) obtained the fusion gene with the signal peptide Mels gene, and cloned it into the pMD 18 vector. The constructed fusion gene plasmids were respectively named: pMOSE2-1, pMOSE2-2. details as follows:

Optimized SM strain fusion gene 1: Mels-optiSE2-1 (abbreviated as MOSE2-1) (SEQ ID NO: 1)

A BamHI restriction site is added upstream, and a stop codon and HindIII site are added downstream

Optimized SM strain fusion gene 2: Mels-optiSE2-2 (abbreviated as MOSE2-2) (sequence 2)

Add SmaI restriction site upstream, add stop codon and KpnI site downstream

4. Subcloning into Baculovirus Transfer Vectors

The pMOSE2-1 and pFastBacDual were subjected to double enzyme digestion, purified and recovered, the digested MOSE2-1 was connected with the pFastBacDual linear plasmid, transformed and identified, and the obtained new plasmid was named pSMOSE2 (pS indicates a single expression plasmid). Then, pMOSE2-2 and pSMOSE2 were subjected to double enzyme digestion respectively, purified and recovered, the enzyme digestion fragmented MOSE2-2 was connected with the pSMOSE2 linear plasmid, transformed, identified, and the obtained new plasmid was named pDMOSE2 (pD indicates double expression plasmid).

5. Transposition to obtain recombinant bacmid DNA

The bacmid DNA constructed by transforming pSMOSE2 and pDMOSE2 into DH10Bac respectively was named as: bSMOSE2 and bDMOSE2.

6. Acquisition of Recombinant Viruses

Recombinant bacmids bSMOSE2 and bDMOSE2 were transfected into sf9 cells with the transfection reagent Cellfectin respectively, expanded and cultured, and the culture supernatant was collected for PCR identification. Obtained two strains of recombinant virus baculovirus BacVSMOSE2 strain and BacVDMOSE2 strain, the suggested taxonomic names of the two strains are both Spodoptera californica nuclear polyhedrosis virus, and the baculovirus BacVDMOSE2 strain was sent to the hospital on June 01, 2016 Handed over to the General Microorganisms Collection Center of the Chinese Academy of Sciences, No. 1, Beichen West Road, Chaoyang District, Beijing, No. 3, Institute of Microbiology, Chinese Academy of Sciences, and China Microorganisms Collection Center, the preservation number is CGMCC No.12546.

7. Expression Analysis

The identified virus was subcultured and expanded, and the second generation was recorded as P2Stock, and the third generation was recorded as P3Stock. The first three generations were used as seed virus, and the 96h culture supernatant of the fourth generation was taken to coat the plates for ELISA analysis (see Figure 1). The results showed that all recombinant viruses could secrete E2 protein, but the expression level of baculovirus BacVDMOSE2 strain (double expression) was significantly higher than that of BacVSMOSE2 strain (single expression).

Example 2──Preparation of CSFV E2 Recombinant Baculovirus Inactivated Vaccine

The correctly identified strain of CSFV E2 recombinant baculovirus BacVDMOSE2 was amplified to P6 generation with sf9 cells, and the virus titer was determined by TCID ₅₀ method. Use ExpressFive Medium (Invitrogen Company) to suspend High Five insect cells (Invitrogen Company), 27 ° C, 180r/min shaking culture, when the High Five insect cells grow to the logarithmic growth phase, replace the new ExpressFive Medium, adjust the cell The concentration is 2-5×10 ⁶ /ml, the cells are infected with the recombinant baculovirus BacVDMOSE2 strain at 1 MOI dose, and harvested after 96 hours. Centrifuge at 2000r/min for 10min, take the supernatant, conduct SDS-PAGE electrophoresis, and calculate the content of the target product. The inactivator BEI was added to the antigen at a final concentration of 1 mmol/L, and inactivated at 37°C for 48 hours to inactivate the recombinant virus. The expression supernatant was mixed with ISA206 adjuvant at a ratio of 1:1, and the antigen solution adjuvant was homogenized with an emulsifier at 12000 r/min for 10 minutes to prepare a recombinant baculovirus inactivated vaccine of classical swine fever virus E2.

Example 3──Swine Fever Virus E2 Gene Recombinant Baculovirus Inactivated Vaccine Pig Immune Test

Take 20 healthy pigs negative for CSF antibody, 12 of which were immunized with 3 batches of CSFV E2 recombinant baculovirus inactivated vaccine, 4 were immunized with attenuated CSF virus (passage cell source), and the other 4 were the control group In the immunization group, 2 ml of inactivated CSFV E2 recombinant baculovirus vaccine was injected intramuscularly into the back of the ear and neck, and the second immunization was carried out in the same dose and way 21 days after the first immunization. Blood was collected every 7 days during the first immunization and second immunization. Antibody levels were measured by separating serum. The results proved that the growth of the inactivated vaccine was basically the same as that of the live vaccine antibody level, which proved that the immune efficacy was equivalent. (See Figure 2)

Example 4

──Safety test of recombinant baculovirus inactivated vaccine of classical swine fever virus E2 gene

After the swine fever virus E2 recombinant baculovirus inactivated vaccine was immunized with 2 times the dose (4m1/head), no local and systemic adverse reactions such as fever, diarrhea, loss of appetite, and injection local swelling were found.

Example 5

——Construction of recombinant baculovirus BacVSMOCE2 strain and BacVDMOCE2 strain

According to the same principle of constructing the recombinant baculovirus BacVDMOSE2 strain of classical swine fever virus E2 in the present invention, the present invention simultaneously constructs a recombinant baculovirus containing the E2 gene of classical classical swine fever virus (strain C).

1. Synthesis of the optimized sequence of the E2 gene of classical swine fever rabbitization attenuated virus (C strain)

The sequence of the E2 gene of CSF attenuated rabbit (C strain) was optimized, and the optimized nucleotide sequence of the E2 gene of C strain was obtained by artificial synthesis technology, cloned into the pMD 18 vector, and named: pMD-CE2.

2. Fusion of classical swine fever attenuated E2 gene and signal peptide sequence

Extract the pMD-CE2 plasmid, add appropriate restriction sites through primers, amplify CE2 and the signal peptide Mels gene by PCR technology, and then obtain the fusion gene with the signal peptide Mels gene by overlap extension PCR technology, and clone it into the pMD 18 vector. The constructed fusion gene plasmids were respectively named: pMOCE2-1, pMOCE2-2. details as follows:

Optimized C strain fusion gene 1: Mels-optiCE2-1 (abbreviated as MOCE2-1) (SEQ ID NO: 3)

A BamHI restriction site is added upstream, and a stop codon and HindIII site are added downstream

Optimized C strain fusion gene 2: Mels-optiCE2-2 (abbreviated as MOCE2-2) (SEQ ID NO: 4)

Add SmaI restriction site upstream, add stop codon and KpnI site downstream

4. Subcloning into Baculovirus Transfer Vectors

The pMOCE2-1 and pFastBacDual were subjected to double enzyme digestion, purified and recovered, the enzyme digested MOCE2-1 was connected with the pFastBacDual linear plasmid, transformed, identified, and the obtained new plasmid was named pSMOCE2 (pS indicates a single expression plasmid). Then, pMOCE2-2 and pSMOCE2 were subjected to double enzyme digestion respectively, purified and recovered, the enzyme digestion fragmented MOCE2-2 was connected with the pSMOCE2 linear plasmid, transformed, identified, and the new plasmid obtained was named pDMOCE2 (pD indicates double expression plasmid).

5. Transposition to obtain recombinant bacmid DNA

The bacmid DNA constructed by transforming pSMOCE2 and pDMOCE2 into DH10Bac respectively was named as: bSMOCE2 and bDMOCE2.

6. Acquisition of Recombinant Viruses

Recombinant bacmids bSMOCE2 and bDMOCE2 were transfected into sf9 cells with the transfection reagent Cellfectin respectively, expanded and cultured, and the culture supernatant was collected for PCR identification. The two recombinant viruses were named as baculovirus BacVSMOCE2 strain and BacVDMOCE2 strain respectively, and the proposed taxonomic names were both S. californica nuclear polyhedrosis virus.

7. Expression Analysis

The identified virus was subcultured and expanded, and the second generation was recorded as P2Stock, and the third generation was recorded as P3Stock. The first three generations were used as seed virus, and the 96h culture supernatant of the fourth generation was taken to coat the plates for ELISA analysis (see Figure 1). The results showed that both the recombinant virus baculovirus BacVSMOCE2 strain and the baculovirus BacVDMOCE2 strain could secrete E2 protein, and both could be used to produce the recombinant baculovirus inactivated vaccine of classical swine fever virus E2, but the expression level of the baculovirus BacVDMOCE2 strain (double expression) Significantly higher than the BacVSMOCE2 strain (single expression) 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.