Classical swine fever virus subunit vaccine and preparation method thereof

Abstract

The invention provides a classical swine fever virus subunit vaccine and a preparation method thereof, the classical swine fever virus subunit vaccine is based on an E2 protein gene and a GP67 signal peptide gene of a CSFV 1.1 strain, a recombinant expression vector is constructed through gene cloning, a classical swine fever virus E2 recombinant protein is obtained through expression of an insect baculovirus system, and the classical swine fever virus subunit vaccine is obtained. And the recombinant protein is combined with a colloidal manganese adjuvant to prepare the swine fever virus subunit vaccine. The hog cholera virus E2 recombinant protein is close to natural E2 protein in structural function, high in reduction degree, high in expression quantity and suitable for vaccine production and application; the vaccine can generate specific humoral immunity and relatively strong cellular immunity, and is beneficial to removal of CSFV in an animal body; meanwhile, the vaccine can be distinguished from attenuated vaccine strain immunization or wild strain infection at the nucleic acid level, can be used for preventing and purifying classical swine fever in an intensive pig farm, and has a considerable application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of veterinary biological products, and specifically relates to a swine fever virus subunit vaccine and a preparation method thereof. Background Art

[0002] Classical swine fever (CSF) is an important infectious disease caused by classical swine fever virus (CSFV), which is an RNA virus. Vaccination is currently the main means of controlling classical swine fever, but traditional vaccines on the market cannot use serological methods to distinguish between vaccine-immunized animals and wild-virus-infected animals, and cannot solve the problem of major animal disease purification. In order to meet the demand for future classical swine fever vaccines, the development of new vaccines is particularly important. Recombinant subunit vaccines have obvious advantages, are safe and efficient, and can be produced on a large scale. They have been favored by the market in recent years. The four structural proteins of classical swine fever are C, Erns, E1, and E2, and E2 is the main antigen that induces the production of neutralizing antibodies and has the highest immunogenicity. Therefore, the study of E2 protein has also become one of the keys to the prevention and treatment of classical swine fever. E2 protein is an envelope glycoprotein, a type I transmembrane protein, has strong immunogenicity, and can induce the body to produce specific antibodies IgG, so E2 protein is usually selected when preparing subunit vaccines. A prominent advantage of the Classical Swine Fever E2 protein subunit vaccine is that it can effectively distinguish between wild strain infection and vaccine immunity, and there is no risk of reverse virulence. These characteristics help to thoroughly control and purify Classical Swine Fever.

[0003] However, in the preparation process of the existing recombinant subunit vaccine of the E2 protein of the classical swine fever virus, there are problems such as poor restoration of the E2 protein structure and function, low protein expression, large amount of antigen used in vaccine preparation, and unsatisfactory immune protection effect of the vaccine. The production cost is high and it is even difficult to use it in large-scale production. Summary of the invention

[0004] In view of the defects of the above-mentioned prior art, the object of the present invention is to provide a swine fever virus subunit vaccine with high protein structure and function restoration, high expression level and good immune protection effect and a preparation method thereof.

[0005] The technical solution adopted by the present invention is as follows:

[0006] A classical swine fever virus subunit vaccine comprising classical swine fever virus E2 recombinant protein and colloidal manganese adjuvant;

[0007] The mass ratio of the classical swine fever virus E2 recombinant protein to manganese in the colloidal manganese adjuvant is 1:5-10.

[0008] Preferably, the colloidal manganese adjuvant is a manganese nanoparticle aqueous adjuvant, and the preparation of the manganese nanoparticle aqueous adjuvant comprises the following steps:

[0009] S1, MnCl2, NAOH and Na3PO4 are mixed and stirred continuously for 6-8 h to generate manganese salt particles;

[0010] S2, centrifugation to collect particles in suspension;

[0011] S3, suspending the particles in physiological saline to prepare the manganese nanoparticle aqueous adjuvant.

[0012] Preferably, in the mixed solution of MnCl2, NAOH and Na3PO4 in step S1, Mn 2+ The concentration of OH was 10 mM. - The concentration is 6-10 mM, PO4 3- The concentration is 5-6 mM;

[0013] And / or, the manganese content in the prepared colloidal manganese adjuvant is 2-5 mg / mL.

[0014] Preferably, the nucleotide sequence encoding the classical swine fever virus E2 recombinant protein is obtained by codon optimization of the E2 protein gene of the CSFV 1.1 strain, and the optimized nucleotide sequence is SEQ ID NO.1.

[0015] Preferably, the nucleotide sequence encoding the classical swine fever virus E2 recombinant protein includes a GP67-E2 gene fragment synthesized based on the optimized E2 protein gene and the GP67 signal peptide gene, and the nucleotide sequence of the GP67 signal peptide gene is SEQ ID NO.2.

[0016] Preferably, the classical swine fever virus E2 recombinant protein is expressed by an insect baculovirus system.

[0017] Based on the same invention purpose, a method for preparing the above-mentioned swine fever virus subunit vaccine is also provided, comprising the following steps:

[0018] S1, synthesizing the GP67-E2 gene fragment based on the E2 protein gene and GP67 signal peptide gene of the codon-optimized CSFV 1.1 strain;

[0019] S2, clone the GP67-E2 gene fragment into the baculovirus expression vector pFastBac1 to construct the recombinant expression vector pFastBac-GP67-E2;

[0020] S3, transforming the recombinant expression vector pFastBac-GP67-E2 into competent cells, culturing, extracting, and identifying to obtain a recombinant pFastBac-GP67-E2 plasmid;

[0021] S4, transfecting the recombinant pFastBac-GP67-E2 plasmid into sf9 cells, culturing the P1 virus and then transfecting the virus into sf9 cells to obtain the P2 virus, continuing the infection until the P3 virus is obtained, collecting the culture supernatant, purifying and concentrating, and obtaining the recombinant protein of classical swine fever virus E2.

[0022] Preferably, the method further comprises step S5, mixing the classical swine fever virus E2 recombinant protein with a colloidal manganese adjuvant to prepare the classical swine fever virus subunit vaccine.

[0023] Preferably, the nucleotide sequence of the recombinant expression vector pFastBac-GP67-E2 is SEQ ID NO.3.

[0024] Preferably, the mass ratio of the classical swine fever virus E2 recombinant protein to the colloidal manganese adjuvant is 1:5-10.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] 1. The swine fever virus subunit vaccine of the present invention, by adding colloidal manganese adjuvant, can effectively improve the immune effect of the swine fever virus subunit vaccine, obtain a stronger immune protection effect than conventional adjuvants, produce a higher antibody concentration, and also produce stronger cellular immunity.

[0027] 2. The classical swine fever virus E2 recombinant protein of the present invention is modified and folded by optimizing the codons of the E2 protein gene and adding the GP67 signal peptide, and expressing it using an insect baculovirus expression system. The function and structure of the classical swine fever virus E2 recombinant protein are closer to the natural E2 protein, the degree of reduction is high, and the amount used in the preparation of the vaccine is smaller; and the classical swine fever virus E2 recombinant protein has a high expression level, is suitable for production and application, and reduces the cost of vaccine production. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0029] Figure 1 The peak graph of classical swine fever virus E2 recombinant protein purified by ÄKTA purifier is shown in Figure 1. FT is the effluent, W is the washing solution, and E is the elution solution.

[0030] Figure 2 This is the SDS-PAGE verification result of the purified classical swine fever virus E2 recombinant protein; wherein, IN is the stock solution, FT is the effluent, W is the washing solution, and E is the elution solution.

[0031] Figure 3 This is the final concentration result of the purified recombinant protein of classical swine fever virus E2.

[0032] Figure 4 The blocking rate results of the rabbit ELISA competition method in Example 2; wherein, 1-5 are E2 recombinant subunit vaccine groups, AC are commercial swine fever live vaccine groups, and ad are blank control groups.

[0033] Figure 5 The results of the changes in body temperature of rabbits before and after virus attack in Example 2; among them, 1-5 are E2 recombinant subunit vaccine groups, AC are commercial swine fever live vaccine groups, and ad are blank control groups.

[0034] Figure 6 This is the result of indirect ELISA detection of mouse serum IgG level in Example 3.

[0035] Figure 7 These are the results of double antibody sandwich ELISA for detecting the levels of IL-4 and IFN-γ in mouse serum in Example 3; wherein, Control is a blank control group, and compared with the blank control group, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P < 0.0001.

[0036] Figure 8 The results of mouse spleen lymphocyte proliferation experiment in Example 3; wherein, Control is a blank control group, and compared with the blank control group, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P < 0.0001. DETAILED DESCRIPTION

[0037] In the following, only some exemplary embodiments are briefly described. As those skilled in the art will appreciate, the described embodiments may be modified in various ways without departing from the spirit or scope of the present invention. Therefore, the drawings and descriptions are considered to be exemplary and non-restrictive in nature.

[0038] The embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0039] Example 1

[0040] Expression and purification of recombinant protein of classical swine fever virus E2

[0041] The E2 protein gene of CSFV 1.1 strain (HVRI of Shimen strain of classical swine fever virus numbered AY775178.2:2441-3559 in the NCBI database) was selected and codon optimized to obtain an optimized E2 protein gene fragment, the nucleotide sequence of which is SEQ ID NO.1.

[0042] Based on the optimized E2 protein gene fragment and GP67 signal peptide gene, the expression gene fragment GP67-E2 of E2 recombinant protein and GP67 signal peptide is synthesized, wherein the nucleotide sequence of GP67 signal peptide gene is SEQ ID NO.2.

[0043] The GP67-E2 gene fragment was cloned into BamH Ⅰ and Xho Ⅰ of the baculovirus expression vector pFastBac1 by gene cloning to construct a recombinant expression vector pFastBac-GP67-E2. The nucleotide sequence of the recombinant expression vector pFastBac-GP67-E2 includes BamH I-GP67-EcoRI-E2-Sal I-6His-Xho I connected in sequence, and the specific nucleotide sequence is SEQ ID NO.3.

[0044] The recombinant expression vector pFastBac-GP67-E2 was ligated and transformed into DH5α competent cells, and the recombinant pFastBac-GP67-E2 plasmid was extracted after culture.

[0045] The extracted recombinant pFastBac-GP67-E2 plasmid was identified. After successful identification, the recombinant pFastBac-GP67-E2 plasmid was transfected into insect cells.

[0046] Specifically, the extracted recombinant pFastBac-GP67-E2 plasmid is transfected into sf9 cells to obtain P1 virus, the P1 virus is infected into sf9 cells to obtain P2 virus, the P2 virus is further infected into sf9 cells to obtain P3 virus, the culture supernatant of the infecting cells is collected, concentrated using a tangential flow ultrafiltration system, purified using an ÄKTA purifier fully automatic chromatography instrument (Ni-NTA vertical gravity column can also be used for purification), and then ultrafiltration and concentration are performed to obtain the classical swine fever virus E2 recombinant protein. After SDS-PAGE verification, the concentration of the classical swine fever virus E2 recombinant protein is detected by dialysis and set aside.

[0047] By adding the GP67 signal peptide gene to the E2 protein gene, the protein expression can be effectively increased, and the secretion of the expressed protein outside the cell can be promoted. The insect baculovirus system is used to modify and fold the expressed protein, making the classical swine fever virus E2 recombinant protein closer to the natural protein and having a higher degree of reduction; combined with the use of the GP67 signal peptide gene, the problem of low expression in the baculovirus expression system is effectively solved. Therefore, the classical swine fever virus E2 recombinant protein obtained by this method has the characteristics of high expression, high degree of reduction, good immunogenicity, and is suitable for vaccine production and application.

[0048] See the peak graph of the above ÄKTA purifier purified classical swine fever virus E2 recombinant protein for details. Figure 1 As shown; SDS-PAGE verification results refer to Figure 2 As shown; the final concentration of the purified recombinant protein of classical swine fever virus E2 is as shown Figure 3 As shown. Figure 3 It can be seen that the band size of the purified classical swine fever virus E2 recombinant protein is correct, there are no other impurity bands, and the protein purity is high.

[0049] Example 2

[0050] Challenge experiment of preparing subunit vaccine with recombinant protein of classical swine fever virus E2

[0051] (1) Preparation of immunogen

[0052] The classical swine fever virus E2 recombinant protein (antigen) prepared in Example 1 was mixed and emulsified with the commercially available ISA 201 VG adjuvant (purchased from Seppic, France) at a ratio of 50:50 (w / w). The vaccine was prepared with 40 μg of classical swine fever virus E2 recombinant protein antigen per vial, that is, the antigen amount of each vaccine was 40 μg / vaccine.

[0053] (2) Group immunization and virus challenge of experimental rabbits

[0054] Twelve New Zealand white rabbits were randomly divided into three groups: blank control group (4 rabbits); E2 recombinant subunit vaccine group (5 rabbits); commercial swine fever live vaccine group (3 rabbits).

[0055] The vaccine was injected intramuscularly or subcutaneously. The blank control group was injected with 0.1 mL of saline solution; the E2 recombinant subunit vaccine group was injected with 0.1 mL of the vaccine prepared with the above pestivirus E2 recombinant protein and ISA 201 VG adjuvant (40 μg); the commercial swine fever live vaccine group was injected with 0.1 mL of a 150-fold diluted swine fever live vaccine (a live attenuated bacterial vaccine, the strain is C strain spleen and lymphotoxin). Among them, the E2 recombinant subunit vaccine group was immunized for the first time on the first day and the second time on the 21st day; the commercial swine fever live vaccine group was immunized once on the 21st day; the blank control group was injected with sterile saline solution on the 1st and 21st days.

[0056] The three groups of animals were challenged with swine fever spleen virus on the 49th day after immunization. The virus was diluted with sterile saline to 1 portion / mL and injected into the rabbits through the ear vein at a dose of 1 mL / rabbit.

[0057] (3) Serum antibody level detection

[0058] Starting from day 0, 2 mL of blood was collected from the rabbit ears every week until day 56. The whole blood was placed in a 37°C constant temperature water bath for 1 hour, kept at 4°C overnight, and centrifuged at 3000 rpm for 10 minutes at 4°C the next day. The supernatant was collected and stored in aliquots at -20°C. Serum antibodies were detected using the ELISA competitive method.

[0059] The results of the rabbit ELISA competitive blocking rate are shown in Figure 4 shown.

[0060] The blocking rate is used to evaluate the concentration of the tested antibody, reflecting the ability of the antibody in the tested sample to bind to the antigen. In the application of swine fever antibody detection, the blocking rate is directly related to the judgment of the antibody concentration. Generally, the higher the blocking rate, the higher the antibody concentration in the sample. Figure 4 The results showed that the blocking rate of the E2 recombinant subunit vaccine group increased faster over time, reached the maximum in a shorter time, and was significantly higher than that of the commercial live swine fever vaccine group, while the blocking rate of the blank control group did not change significantly. This shows that the E2 recombinant subunit vaccine group has a better immune effect than the commercial live swine fever vaccine group.

[0061] (4) Body temperature measurement after virus attack

[0062] Rectal temperature was measured starting one day before the challenge and recorded every 6-12 hours after the challenge to detect typical fever in rabbits. The normal body temperature of rabbits is generally maintained at 38.5℃~39.5℃.

[0063] The results of the changes in body temperature of rabbits in each group before and after the virus attack are as follows Figure 5 shown.

[0064] The judgment criteria are divided into four standard levels: (A) Stereotyped fever reaction (++): The body temperature rises significantly within a period of time after the challenge, with at least three temperatures exceeding the normal body temperature by more than 1°C and the abnormal body temperature remains for 18 to 36 hours. (B) Mild fever reaction (+): The body temperature rises significantly within a period of time after the challenge, and two temperatures exceed the normal body temperature by more than 0.5°C, and the abnormal body temperature remains for 12 to 36 hours. (C) Suspected reaction (±): The body temperature fluctuates for a period of time after the challenge, but the duration is short, usually within 12 hours. (D) No reaction (-): The body temperature continues to remain within the normal temperature level.

[0065] according to Figure 5 The results showed that the body temperature of rabbits in the E2 recombinant subunit vaccine group (1-5) and the commercial swine fever live vaccine group (AC) was within the normal range after the virus attack, and there was no significant increase, which was judged as no reaction (-); the body temperature of rabbits in the blank control group rose significantly after the virus attack, and the temperature exceeded the normal body temperature by more than 1°C for more than three times and the abnormal temperature was maintained for 18 to 36 hours, which was judged as stereotyped heat reaction (++). This shows that the virus attack was successful, and the rabbits immunized with the E2 recombinant subunit vaccine (1-5) and the commercial swine fever live vaccine (AC) had a good immune protection effect.

[0066] Example 3

[0067] Immunological evaluation of classical swine fever virus subunit vaccine adjuvanted with colloidal manganese in mice

[0068] (1) Preparation of colloidal manganese adjuvant and immunogen

[0069] Colloidal manganese adjuvant (MnJ adjuvant): Mix MnCl2, NAOH and Na3PO4 to control the Mn content in the mixed solution. 2+ The concentration of OH was 10 mM. - The concentration is 6-10 mM, PO4 3- The concentration of Mn was 5-6 mM. The Mn salt particles were generated by continuous stirring at 500 rpm for 6-8 h. Then, the particles in the suspension were collected by centrifugation at 1000 × g for 30 min. The collected particles were suspended in physiological saline to control the concentration of Mn 2+ The concentration of the manganese nanoparticles is 2-5 mg / mL to obtain the desired colloidal manganese adjuvant, which is an aqueous manganese nanoparticle adjuvant.

[0070] ISA 201 VG adjuvant vaccine: 100 μg of classical swine fever virus E2 recombinant protein was mixed with ISA 201 VG adjuvant (purchased from Seppic, France) at a ratio of 50:50 (w / w) to prepare the vaccine.

[0071] Aluminum hydroxide adjuvant vaccine: Shake the commercially available bottled injection alum before use, then drop the injection alum into the swine fever virus E2 recombinant protein immunogen solution and stir continuously to make the final volume ratio of injection alum to immunogen be 1:3 (can be 1:1-1:3). Continue stirring for 30 minutes after adding the injection alum to allow the injection alum to effectively adsorb the antigen and prepare the vaccine.

[0072] β-Glucan adjuvant vaccine: 100 μg of classical swine fever virus E2 recombinant protein was mixed with 2% β-glucan aqueous solution to prepare the vaccine.

[0073] MnJ (colloidal manganese) adjuvant vaccine: 100 μg of classical swine fever virus E2 recombinant protein was mixed with 5 times the amount of colloidal manganese adjuvant (measured by weight of manganese ions), mixed thoroughly and allowed to stand at room temperature, then physiological saline was added to control the adjuvant concentration to 1 mg / mL, and the vaccine was prepared by mixing again. The amount of colloidal manganese adjuvant added can be 5 to 10 times the amount of classical swine fever virus E2 recombinant protein, and a better immune effect can be obtained.

[0074] The mixture of antigen (recombinant protein of classical swine fever virus E2) and adjuvant is ready for use, and immunization is completed within 30 minutes. For each injection, 100 μg of immunogen (recombinant protein of classical swine fever virus E2) is used per mouse.

[0075] (2) Immunization and sampling of BALB / c mice

[0076] Thirty-six mice were randomly divided into 6 groups, with 6 mice in each group: blank group (Control), aluminum hydroxide adjuvant group (Al(OH)2), β-glucan adjuvant group (β-Glu), MnJ adjuvant group (MnJ), and ISA 201 VG adjuvant group, which were further divided into primary immunization group (ISA 201-1) and secondary immunization group (ISA 201-2) for intraperitoneal or subcutaneous multiple-point immunization.

[0077] (3) Serum specific antibody level detection

[0078] Starting from the 0th day, the mice were tail-cut and blood was collected 100 μL every week until the 35th day. The whole blood was taken out and placed in a constant temperature water bath at 37℃ for 1 hour, and then kept at 4℃ overnight. The next day, it was centrifuged at 12000 rpm at 4℃ for 15 minutes, and the supernatant was taken and stored in aliquots at -20℃. The indirect method was used to detect the specific antibody level in the serum. The test results are shown in Figure 6 shown.

[0079] according to Figure 6The results showed that the IgG antibody level of the Control group remained unchanged from 0 to 4 weeks, and the IgG antibody levels of the MnJ, Al (OH) 2, ISA 201-2, ISA 201-1 and β-Glu groups began to increase significantly after 2 weeks, and the IgG antibody level of the MnJ group was significantly higher than that of the other groups, indicating that the vaccine successfully stimulated the immune system response of the mice, produced the required antibodies, and played an immune protection effect, and the MnJ group had the best immune protection effect. It further shows that the addition of colloidal manganese adjuvant to the recombinant protein of the swine fever virus E2 can have better immune protection efficacy than adding conventional adjuvants.

[0080] (4) Serum cytokine level detection

[0081] Commercially available double antibody sandwich ELISA kits were used for detection. Mouse interleukin 4 (IL-4) or interferon gamma (IFN-γ) ELISA kits and serum samples were equilibrated at room temperature for 15-30 min before use.

[0082] Test results such as Figure 7 shown.

[0083] according to Figure 7 The results showed that the levels of IL-4 and IFN-γ in the serum of mice in the MnJ, Al(OH)2, ISA 201-2, ISA 201-1 and β-Glu groups were higher than those in the Control group. Among them, the levels of IL-4 and IFN-γ in the serum of mice in the β-Glu group did not increase significantly; the levels of IFN-γ in the serum of mice in the MnJ and ISA 201-2 groups increased most significantly; the level of IL-4 in the serum of mice in the MnJ group was not the highest, but it was the same as that in the Al(OH)2 and ISA 201-1 groups, reaching a significantly increased level; indicating that the mice in the MnJ group had stronger cellular immunity.

[0084] (5) Splenic lymphocyte proliferation assay

[0085] The spleen lymphocytes isolated from the spleens of mice immunized with 6 groups of different adjuvant vaccines were diluted to a concentration of 1×10 6 Cell / mL. At the same time, ConA was also diluted to 10 µg / mL using complete medium. 100 µL of lymphocytes were added to each well of a 96-well cell culture plate, followed by 100 µL of ConA, and sample duplicate wells and control wells were set. Cultured in a 37°C, 5% CO2 incubator for 66-72 h. Afterwards, 20 µL of CCK-8 solution was added to each well, and cultured for another 4 h under the same conditions. The OD value was measured at a wavelength of λ=450 nm using an enzyme reader, and the stimulation index (SI) value was calculated as a parameter for determining the degree of lymphocyte transformation.

[0086] Stimulation index (SI) = (OD_450 nm (test well)) / (OD_450 nm (control well)).

[0087] Mouse spleen lymphocyte proliferation experiment results Figure 8 shown.

[0088] according to Figure 8 The results showed that the proliferation of spleen lymphocytes in the MnJ group of mice was the most significant, indicating that colloidal manganese adjuvant has the most significant effect on promoting the proliferation of mouse spleen lymphocytes.

Claims

1. A swine fever virus subunit vaccine, characterized in that: It includes recombinant protein of classical swine fever virus E2 and colloidal manganese adjuvant; The mass ratio of the classical swine fever virus E2 recombinant protein to manganese in the colloidal manganese adjuvant is 1:5-10.

2. The swine fever virus subunit vaccine according to claim 1, characterized in that The colloidal manganese adjuvant is a manganese nanoparticle aqueous adjuvant, and the preparation of the manganese nanoparticle aqueous adjuvant comprises the following steps: S1, MnCl2, NAOH and Na3PO4 are mixed and stirred continuously for 6-8 h to generate manganese salt particles; S2, centrifugation to collect particles in suspension; S3, suspending the particles in physiological saline to prepare the manganese nanoparticle aqueous adjuvant.

3. The swine fever virus subunit vaccine according to claim 2, characterized in that: In the mixed solution of MnCl2, NAOH and Na3PO4 in step S1, Mn 2+ The concentration of OH was 10 mM. - The concentration is 6-10 mM, PO4 3- The concentration is 5-6 mM; And / or, the manganese content in the prepared colloidal manganese adjuvant is 2-5 mg / mL.

4. The classical swine fever virus subunit vaccine according to any one of claims 1 to 3, characterized in that The nucleotide sequence encoding the classical swine fever virus E2 recombinant protein is obtained by codon optimization of the E2 protein gene of the CSFV 1.1 strain, and the optimized nucleotide sequence is SEQ ID NO.

1.

5. The swine fever virus subunit vaccine according to claim 4, characterized in that The nucleotide sequence encoding the classical swine fever virus E2 recombinant protein includes a GP67-E2 gene fragment synthesized based on the optimized E2 protein gene and the GP67 signal peptide gene, and the nucleotide sequence of the GP67 signal peptide gene is SEQ ID NO.

2.

6. The swine fever virus subunit vaccine according to claim 5, characterized in that The classical swine fever virus E2 recombinant protein is expressed through an insect baculovirus system.

7. The method for preparing a swine fever virus subunit vaccine according to any one of claims 1 to 6, characterized in that: The following steps are involved: S1, synthesizing the GP67-E2 gene fragment based on the E2 protein gene and GP67 signal peptide gene of the codon-optimized CSFV 1.1 strain; S2, clone the GP67-E2 gene fragment into the baculovirus expression vector pFastBac1 to construct the recombinant expression vector pFastBac-GP67-E2; S3, transforming the recombinant expression vector pFastBac-GP67-E2 into competent cells, culturing, extracting, and identifying to obtain a recombinant pFastBac-GP67-E2 plasmid; S4, transfecting the recombinant pFastBac-GP67-E2 plasmid into sf9 cells, culturing the P1 virus and then transfecting the virus into sf9 cells to obtain the P2 virus, continuing the infection until the P3 virus is obtained, collecting the culture supernatant, purifying and concentrating, and obtaining the recombinant protein of classical swine fever virus E2.

8. The method for preparing a swine fever virus subunit vaccine according to claim 7, characterized in that: The method further comprises step S5, mixing the classical swine fever virus E2 recombinant protein with a colloidal manganese adjuvant to prepare the classical swine fever virus subunit vaccine.

9. The method for preparing a swine fever virus subunit vaccine according to claim 7, characterized in that: The nucleotide sequence of the recombinant expression vector pFastBac-GP67-E2 is SEQ ID NO.

3.

10. The method for preparing a swine fever virus subunit vaccine according to claim 8, characterized in that: The mass ratio of the classical swine fever virus E2 recombinant protein to the colloidal manganese adjuvant is 1:5-10.

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