CN111000995A

CN111000995A - Triple inactivated vaccine for pigs and preparation method and application thereof

Info

Publication number: CN111000995A
Application number: CN201911379890.0A
Authority: CN
Inventors: 梁宛楠; 孙丹; 赵晓春; 闫妍; 刘鑫莹; 孙晓峰; 刘艳昭; 李琳; 杨大伟; 蒋迪
Original assignee: Harbin Pharmaceutical Group Bio Vaccine Co ltd
Current assignee: Harbin Pharmaceutical Group Bio Vaccine Co ltd
Priority date: 2019-12-27
Filing date: 2019-12-27
Publication date: 2020-04-14

Abstract

The invention discloses a triple inactivated vaccine for pigs and a preparation method and application thereof. The triple inactivated vaccine for pigs comprises: recombinant baculovirus expressing type O FMDV VP1 gene, porcine Seneca virus, H3N2 subtype swine influenza virus and immunological adjuvant. The invention also discloses a method for preparing the triple inactivated vaccine for pigs, which comprises the following steps: (1) amplifying the recombinant baculovirus expressing the O-type FMDV VP1 gene and then inactivating the recombinant baculovirus; (2) mixing inactivated recombinant baculovirus solution expressing O-type FMDV VP1 gene, inactivated swine Senecan virus solution and H3N2 subtype swine influenza virus to obtain a water phase; (3) heating the immunologic adjuvant to obtain an oil phase; (4) adding the oil phase into the water phase, mixing, and emulsifying. The immune protection efficacy test result proves that the triple inactivated vaccine for the pigs prepared by the invention can effectively prevent and treat O-type foot-and-mouth disease virus, porcine Seneca virus and H3N2 subtype swine influenza virus at the same time.

Description

Triple inactivated vaccine for pigs and preparation method and application thereof

Technical Field

The invention relates to a triple inactivated vaccine for pigs, in particular to a triple inactivated vaccine for a foot-and-mouth disease baculovirus vector, a swine Seneca virus and an H3N2 subtype swine influenza virus and a preparation method thereof, belonging to the field of triple inactivated vaccines for pigs.

Background

The type O foot-and-mouth disease virus is one of the members of the foot-and-mouth disease virus genus and the picornaviridae family. Of all seven serotypes of foot-and-mouth disease virus, type O is the most prevalent and widespread throughout the world, reported in northern africa, eastern europe, whole south america, and most regions of asia, and seriously threatens the development of the animal husbandry worldwide. The inactivated vaccine has the defects of high equipment safety requirement, complex process, high cost, incomplete inactivation of a strain, danger of virus dispersion and the like in the preparation process, so that the baculovirus vector vaccine is considered as a candidate vaccine with the most potential in the field of foot-and-mouth disease virus vaccine research, and the virus-like particles with the virus epitope are synthesized and expressed by using a gene recombination technology, can induce animals to generate immunity which is approximately natural virus infection, and are always highly regarded.

The seneca valley virus was first isolated by researchers from cell culture contaminants of human embryonic retinal cells per.c6 and was thought to originate from bovine serum or porcine trypsin used by cultured cells. In 2015, brazilian scholars detected the presence of SVA whole genome from the vacuolar fluid and serum of pigs with vesicular disease and considered that SVV infection was associated with idiopathic vesicular disease in pigs. Cases of SVV infection in pigs have subsequently been reported in several countries around the world, such as the United states, Brazil, Canada, China, Thailand. SVV can cause infection of herds at different ages, and is clinically manifested by blisters and ulcers in the hooves, mouths and noses, and death of infected newborn piglets. Seneca virus currently has no commercial vaccine production.

Influenza virus belongs to the family of orthomyxoviridae, and was first discovered in 1918 in the united states to cause an acute, febrile, highly contagious respiratory infectious disease. The high-incidence seasons of the swine influenza are mostly in the late autumn, the cold winter and the early spring, and the swine influenza is implosively developed in a short time. Typical clinical symptoms of pigs infected with SIV are cough, sneeze, watery nasal discharge, high fever, anorexia, mental depression, exhaustion, dyspnea, delayed slaughter, reproductive disturbance and the like, and the typical clinical symptoms are characterized by strong infectivity, high morbidity and low mortality, but can seriously affect the growth performance of the pigs and delay slaughter time; meanwhile, the swine influenza is very easy to be mixed with other respiratory diseases or secondarily infected to develop a swine respiratory disease syndrome, so that the death rate is increased suddenly, and great harm is caused to the swine industry.

So far, a triple inactivated vaccine for pigs, which can effectively prevent and treat O-type foot-and-mouth disease virus, porcine Seneca Valley virus and influenza virus at the same time, is lacked.

Disclosure of Invention

One of the purposes of the invention is to provide a triple inactivated vaccine for pigs for effectively preventing and treating O-type foot-and-mouth disease virus, porcine Seneca Valley virus and influenza virus simultaneously

The second purpose of the invention is to provide a method for preparing the triple inactivated vaccine for pigs;

the above object of the present invention is achieved by the following technical solutions:

a triple inactivated vaccine for pigs, comprising: recombinant baculovirus expressing type O FMDV VP1 gene, porcine Seneca virus, H3N2 subtype swine influenza virus and immunological adjuvant.

The construction method of the recombinant baculovirus expressing the O-type FMDV VP1 gene comprises the following steps: .

(1) Cloning the O-type FMDV VP1 gene to a pFastBac I vector to obtain a recombinant plasmid pFB-FMDV-VP 1; (2) constructing a recombinant shuttle rod plasmid rBacmid-FMDV-vp1 by using a recombinant plasmid pFB-FMDV-vp 1; (3) transfecting the insect cells with the recombinant shuttle baculovirus plasmid to obtain the recombinant baculovirus expressing the O-type FMDV VP1 gene.

In order to improve the expression efficiency of the O-type FMDV VP1 gene, the O-type FMDV VP1 gene can be codon optimized to improve the expression efficiency in insect host cells; wherein, the original sequence of the O-type FMDV VP1 gene is shown in SEQ ID NO.1, and the sequence after codon optimization is shown in SEQ ID NO. 2; the expression efficiency of the sequence after codon optimization in insect cells is remarkably improved compared with the original sequence.

Wherein, the insect cell is preferably Sf9 cell.

The porcine Seneca virus of the invention is preferably a microorganism with the preservation number as follows: porcine Seneca Virus of CGMCC No. 18851.

The H3N2 subtype influenza virus in the invention is preferably a microorganism with the preservation number of: influenza virus of CGMCC No. 14740.

The invention further provides a method for preparing the triple inactivated vaccine for the pigs, which comprises the following steps:

(1) amplifying the recombinant baculovirus expressing the O-type FMDV VP1 gene and then inactivating the recombinant baculovirus; amplifying the porcine Seneca virus and then inactivating the porcine Seneca virus; (2) mixing the inactivated recombinant baculovirus solution expressing the O-type FMDV VP1 gene, the inactivated porcine epinakai virus solution and the inactivated H3N2 subtype influenza virus solution uniformly to obtain a water phase; (3) heating the immunologic adjuvant to obtain an oil phase; (4) adding the oil phase into the water phase, mixing, and emulsifying.

In order to obtain better effect, in step (2), the inactivated FMDV-vp1 recombinant baculovirus liquid, the porcine Seneca virus liquid and the influenza virus liquid of H3N2 subtype are mixed to ensure that the content of FMDV-vp1 protein in each vaccine is not less than 40 mu g and the content of porcine Seneca virus is not less than 10 mu g^8.0TCID₅₀H3N2 subtype swine influenza virus not less than 10^7.5EID₅₀。

The immunological adjuvant in the invention is preferably Montanide of French Saibox company^TM206 adjuvant; among them, in the step (3), the immunoadjuvant is preferably heated to 30 ℃ to obtain an oil phase.

Controlling the volume ratio of the oil phase to the water phase to be 1:1 in the step (4); wherein, the water phase is firstly added into the emulsification tank to be slowly stirred, then the oil phase adjuvant is slowly added, and after the addition is finished, the stirring is carried out for 30 minutes at the speed of 800r/min, and then the standing is carried out for 30 minutes.

The immune protection efficacy test of the inactivated vaccine shows that the triple inactivated vaccine for the pigs prepared by the invention is used for immunizing the pigs, blood is collected every week after the pigs are immunized, the FMDV antibody level in the pig serum is determined by using a foot-and-mouth disease O type antibody liquid phase blocking ELISA detection kit, the SVA neutralizing antibody level in the serum is determined by using a neutralizing test method, and the HI antibody level of the neutralizing swine influenza virus in the serum is determined by using a neutralizing test hemagglutination inhibition test method. According to the detection results, high-level FMDV antibodies, SVA antibodies and SIA antibodies are simultaneously generated in the serum of all immunized pigs, and the immune protection efficacy detection results prove that the triple inactivated vaccine for pigs prepared by the invention can be used for preventing and treating O-type foot-and-mouth disease virus, swine Seneca virus and H3N2 subtype influenza virus simultaneously.

Drawings

FIG. 1 PCR identification of recombinant baculovirus shuttle vector DNA; 1: DNA molecular marker DL 2000; 2: constructing a recombinant baculovirus shuttle vector; 3: and (5) negative control.

Detailed Description

The invention is further described below in conjunction with specific embodiments, the advantages and features of which will become apparent from the description. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be within the scope of the invention.

Example 1 preparation of triple inactivated vaccine for pigs

1 seed of Vibrio species

The porcine Sendai virus has the microorganism preservation number as follows: CGMCC No. 18851; and (3) classification and naming: a seneca virus; the preservation unit: china general microbiological culture Collection center; the preservation time is 2019, 10 and 29 months; and (4) storage address: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North.

H3N2 subtype swine influenza virus, its microorganism deposit number is: CGMCC No. 14740; the classification nomenclature is: H3N2 subtype swine influenza virus; the preservation unit: china general microbiological culture Collection center; the preservation time is 11 months and 16 days in 2017; and (4) storage address: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North.

2 test method

2.1 preparation of baculovirus of pig foot-and-mouth disease

2.1.1 design and Synthesis of FMDV-vp1 Gene

The O-type FMDV VP1 gene (SEQ ID NO.1) is subjected to codon optimization to obtain an optimized sequence (SEQ ID NO.2), EcoRI and Hind III enzyme cutting sites are added at two ends of the optimized sequence to be cloned to a pFastBac I vector, and then heavy pFB-FMDV-VP1 is formed.

2.1.2 construction and preparation of recombinant shuttle rod plasmid rBacmid-FMDV-vp1

2.1.2.1 construction of recombinant shuttle rods

mu.L of the recombinant plasmid pFB-FMDV-vp1 was added to 200. mu.L and thawed on iceThe DH10Bac competent cells are gently mixed, ice-bathed for 30min, heat-shocked for 45s at 42 ℃, rapidly placed on ice for 2min, added with 900 mu L of S.O.C culture medium, shake-cultured for 4h at 37 ℃ and 220rpm, and then the S.O.C culture medium is used as 10^-1，10^-2，10^-3And uniformly coating 150 mu L of each dilution on an LB selective agar plate, and culturing in an incubator at 37 ℃ for 36-48h in the dark until clear blue-white colonies appear.

2.1.2.2 extraction of recombinant baculovirus shuttle vector DNA

Placing an LB selective plate in a refrigerator at 4 ℃ for 2h to fully develop colonies, taking out the plate to perform blue-white spot screening, selecting large white single colonies scattered on the plate, streaking and inoculating the large white single colonies scattered on the LB selective plate to perform secondary blue-white spot screening, performing overnight culture at 37 ℃, similarly, selecting large white single colonies scattered on the plate to inoculate the large white single colonies to an LB liquid culture medium containing 50 mu g/ml kanamycin, 7 mu g/ml gentamicin and 10 mu g/ml tetracycline, performing shake culture at 220rpm at 37 ℃ for 16 hours, and extracting DNA of the stem particles.

2.1.2.3 PCR identification of recombinant baculovirus shuttle vector DNA

PCR amplification was performed using the extracted recombinant baculovirus shuttle vector DNA as template and FMDV-vp1-R/FMDV-vp1-F primer of Table 1.

TABLE 1 FMDV-vp1-R/FMDV-vp1-F primer sequences

Circulation parameters: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 10min, wherein 30 cycles are set; extension at 72 ℃ for 10 min. The PCR amplified fragment was estimated to be about 630bp in length, and the correct recombinant baculovirus plasmid DNA was verified to be named rBacmid-FMDV-vp 1.

The constructed shuttle vector of the recombinant baculovirus is subjected to PCR identification, and the length of the amplified fragment is 630bp (figure 1). The identification result shows that FMDV-vp1 protein is successfully transformed into DH10Bac competent cells.

And (3) drawing a standard curve according to the measured OD values of the standard substances with different concentrations, taking the OD values as vertical coordinates and the concentrations as horizontal coordinates, and calculating the concentration of FMDV-vp1 protein to be 1.6mg/ml according to a linear regression equation.

2.1.3 transfection of recombinant baculoplasmids into Sf9 cells

Well-selected Sf9 cells were plated on a 6-well plate before transfection, and an appropriate amount of passage Sf9 cells were stained with Trypan blue for about 30 seconds, dropped on a hemocytometer plate, and the cells were counted by observing the cell activity. Spreading cells with cell activity not less than 97% in 6-well plate with 10 cells per well⁶About one cell. Cells attached after 2h (or overnight) and were available for transfection. The transfection procedure was as follows (cell passage, transfection were performed under sterile conditions):

1. preparing two sterilized 1.5ml EP tubes, and subpackaging 0.1ml Sf-900 culture medium without antibiotics and fetal calf serum respectively;

2. mu.l of recombinant bacmids (2. mu.g) and 6. mu.l of transfection reagent were added, mixed gently, and allowed to stand at room temperature for 5 min.

3. Transferring the culture medium containing the transfection reagent into the culture medium containing the bacmid, gently mixing the components uniformly, and standing the mixture for 20min at room temperature; taking out adherent Sf9 cells, discarding the original culture medium, gently washing twice with Sf-900 culture medium without antibiotics and fetal calf serum, and adding 2ml Sf-900 culture medium without antibiotics and fetal calf serum into each hole;

4. the above 209. mu.l of the mixture was dropped uniformly into one cell well of a 6-well plate, and the other well was a normal cell control. And observing cell changes on 3-5 days of transfection.

2.1.4 amplification and titer determination of recombinant baculovirus

Transferring the Sf9 cell suspension with good state to a cell bottle, and inoculating 10 mu l P0 virus solution after the cells adhere to the wall. After inoculation for about 72h, Sf9 cells begin to become diseased, and virus liquid is harvested on day 5 or 6 when the cells become large and round and intracellular granules are very obvious, and is stored in the dark at minus 80 ℃.

2.1.5 determination of FMDV-vp1 protein

The concentration of FMDV-vp1 protein was determined using a Bradford protein quantitative assay kit using a microplate reader method, and the assay was performed strictly as per the instructions.

2.1.6 inactivation and inactivation assay

2.1.6.1 inactivation

Slowly adding β -propiolactone solution into the obtained pig foot-and-mouth disease baculovirus solution to enable the final concentration to be 0.05%, fully and uniformly mixing, inactivating at 4 ℃ for 8 hours, stirring once every two hours, placing the virus solution at 37 ℃ for hydrolysis for 2 hours after inactivation, and storing the inactivated virus solution at 2-8 ℃.

2.1.6.2 inactivation test

And (3) taking the inactivated virus liquid, inoculating the virus liquid to Sf9 cells which form a good monolayer, culturing and observing for 4 days at 27 ℃, repeatedly freezing and thawing for 2 times, conducting blind passage for 2 generations, and observing the pathological condition of the cells.

The inactivated pig foot-and-mouth disease baculovirus is inoculated with SF9 cells and is blind transferred for 2 generations, no pathological changes are generated in the cells, and the result shows that the virus liquid is completely inactivated.

2.2 Swine Sambucus Canitis Virus preparation

2.2.1 viral propagation

Delivering the BHK-21 cell suspension in good state to a cell bottle, inoculating the virus seeds to the monolayer-grown BHK-21 cells according to 5 per mill of the total amount of the culture medium, and placing the cell in a 5% CO atmosphere at 37 DEG C₂Culturing in incubator, adsorbing for 1 hr, adding DMEM cell culture solution with serum content of 2%, and continuing to culture at 37 deg.C with 5% CO₂Culturing in an incubator. After inoculation, the cells are cultured for 40-48 hours, and when the cytopathic effect reaches more than 80%, the cells and cell cultures are harvested. After repeated freeze thawing for 2 times, TCID is used₅₀The method detects the virus titer.

And (3) determining the content of the porcine Sendai virus: inoculating porcine epinakavirus seed virus to BHK-21 cells growing on one side, culturing for 44 hr, harvesting, repeatedly freezing and thawing for 2 times, and measuring virus content to be 10^8.7TCID₅₀/ml。

2.2.2 Virus inactivation and testing

2.2.1.1 Virus inactivation

Slowly adding β -propiolactone solution into the harvested porcine epinakavirus to enable the final concentration to be 0.05%, fully and uniformly mixing, inactivating for 8 hours at 4 ℃, stirring once every two hours, after inactivation, putting the virus liquid at 37 ℃ for hydrolysis for 2 hours, and storing the inactivated virus liquid at 2-8 ℃.

2.2.1.2 Virus inactivation assay

Taking the inactivated antigen solution, inoculating the inactivated antigen solution to BHK-21 cells full of a monolayer according to 1 per mill of the total amount of the culture solution, and culturing for 48 hours at 37 ℃. After freezing and thawing for 2 times, inoculating cells for blind passage for 2 generations, and observing the pathological condition of the cells. The inactivated porcine epinakavirus is inoculated to BHK-21 cells and blind transferred for 2 generations, and the cells have no pathological changes, and the result shows that the virus liquid is completely inactivated.

2.3H3 subtype Swine influenza Virus preparation

2.3.1 viral propagation

Transferring the MDCK cell suspension to a cell bottle, inoculating the virus seeds into full monolayer MDCK cells according to 1% of the total amount of the culture medium, adding TPCK-pancreatin to make the final concentration of the virus seeds be 2.5 mu g/ml, supplementing DMEM cell culture solution with 2% of serum content, and placing the cell culture solution at 37 ℃ in 5% CO₂Culturing in an incubator, culturing the cells for 72 hours after inoculation, and harvesting the cells and cell culture when the cytopathic effect reaches more than 80%. Repeatedly freezing and thawing for 2 times, and using EID₅₀The method detects the virus titer.

2.3.2 viral inactivation

Slowly adding β -propiolactone solution into the harvested swine influenza virus to enable the final concentration to be 0.08%, fully and uniformly mixing, inactivating for 10 hours at 4 ℃, stirring once every two hours, after inactivation, putting the virus liquid at 37 ℃ for hydrolysis for 2 hours, and storing the inactivated virus liquid at 2-8 ℃.

2.3.4 Virus inactivation assay

Taking the inactivated swine influenza virus, inoculating 1 percent of the total amount of the culture solution to full monolayer MDCK cells, and culturing at 37 ℃ for 72 hours. After freezing and thawing for 2 times, inoculating cells for blind passage for 2 generations, and observing the pathological condition of the cells.

2.4 preparation of inactivated vaccine

2.4.1 preparation of the oil phase Montanide from Spanish Coulter^TM206 adjuvant, heated to 30 ℃.

2.4.2 aqueous phase preparation FMDV-vp1 recombinant baculovirus solution, porcine Seneca virus solution and porcine influenza virus were mixed in a certain ratio to make FMDV-vp1 protein content not less than 40 μ g and porcine Seneca virus in each vaccineToxicity is not less than 10^8.0TCID₅₀Swine influenza virus of not less than 10^7.5EID₅₀

2.4.3 the volume ratio of the emulsified aqueous phase to the oil phase is 1: 1. Firstly, adding the water phase into an emulsification tank, slowly stirring, then slowly adding the oil phase adjuvant, stirring for 30 minutes at 800r/min after adding, and standing for 30 minutes.

Test example 1 test of finished product of triple inactivated vaccine for pig

1 sterility test

The triple inactivated vaccine of the foot-and-mouth disease baculovirus, the porcine epinakal virus and the H3 subtype swine influenza virus is tested according to the annex Di of the current Chinese veterinary pharmacopoeia. The result shows that the finished product is qualified by aseptic inspection.

2 safety inspection

5 healthy susceptible pigs of 8 weeks old are taken, 6ml of the pig foot-and-mouth disease baculovirus vector, the pig seneca virus and the H3 subtype swine influenza virus triple inactivated vaccine is inoculated to muscles of each neck, and 7 days of observation are carried out to observe whether adverse reactions exist after the vaccines are inoculated. 5 healthy susceptible pigs were vaccinated with the vaccine, and 5 test pigs were all healthy during the observation period, with no local and systemic adverse reactions.

Test example 2 efficacy test of triple inactivated vaccine for swine

Test method 1

10 healthy susceptible pigs of 8 weeks of age, which are FMDV negative, SVA negative and SIV negative, were screened, and the test pigs were randomly divided into 2 groups of 5 pigs each. Group 1, 3ml of vaccine was administered to the muscles of each neck, and booster immunization was performed 1 time in the same manner 3 weeks after administration. Group 2 was not immunized and served as a blank control.

1.1 FMDV parts

Blood is collected and serum is separated every week after first immunization, and FMDV antibody level in pig serum is determined by foot-and-mouth disease O-type antibody liquid phase blocking ELISA detection kit.

1.2 SVA fraction

Serum was isolated weekly after priming and neutralization test methods were used to determine the level of neutralizing antibodies to Seneca virus in porcine serum.

1.3 SIA fraction

Blood is collected every week after first-time immunization, serum is separated, and H3 subtype swine influenza HI antibody titer in pig serum is determined by a neutralization test method.

2 test results

2.1 FMDV-specific antibody levels

Blood was collected weekly after immunization of pigs, and FMDV antibody levels in pig serum were determined using a foot-and-mouth disease O-antibody liquid blocking ELISA detection kit produced by the landau veterinary institute of china agricultural academy of sciences (table 1). The results showed that the antibody levels in all immunized groups reached the highest at week 4 or 5, up to between 1:512 and 1:1024, and were almost unchanged at week 6 after reaching the highest level, compared to the control group.

TABLE 1 FMDV-specific antibody levels in sera

Note: "-" represents

2.2SVA neutralizing antibody levels

Blood was collected weekly after swine immunization and serum levels of SVA neutralizing antibodies were determined using a neutralization assay (Table 2). The results showed that the antibody levels in all immunized groups reached the highest at week 4 or 5, up to between 1:512 and 1:2048, and were nearly unchanged at week 6 after reaching the highest level, compared to the control group.

TABLE 2 levels of SVA neutralizing antibodies in serum

2.3H3 subtype Swine influenza Virus HI antibody levels

Blood was collected weekly after swine immunization and serum levels of neutralizing swine influenza virus HI antibodies were determined using a neutralization hemagglutination inhibition assay (table 3). The results showed that the antibody levels in all immunized groups were highest at week 5 or 6, up to between 1:640 and 1:2560, compared to the control group.

TABLE 3 serum H3 subtype Swine influenza Virus HI antibody levels

SEQUENCE LISTING

<110> Harbin group biological vaccine Co., Ltd

<120> triple inactivated vaccine for pigs, preparation method and application thereof

<130>HLJ-3002-191203A

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<170>PatentIn version 3.5

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Claims

1. A triple inactivated vaccine for pigs is characterized by comprising: recombinant baculovirus expressing type O FMDV VP1 gene, porcine Seneca virus, H3N2 subtype swine influenza virus and immunological adjuvant.

2. The triple inactivated vaccine for swine as claimed in claim 1, wherein the method for constructing the recombinant baculovirus expressing the type O FMDV VP1 gene comprises:

(1) cloning the O-type FMDV VP1 gene to a pFastBac I vector to obtain a recombinant plasmid pFB-FMDV-VP 1; (2) constructing a recombinant shuttle rod plasmid rBacmid-FMDV-vp1 by using a recombinant plasmid pFB-FMDV-vp 1; (3) and (3) transfecting the insect cells with the recombinant shuttle baculovirus plasmid to obtain the recombinant baculovirus expressing the O-type FMDV VP1 gene.

3. The triple inactivated vaccine for pigs according to claim 2, wherein the O-type FMDV VP1 gene is a codon optimized gene, and the nucleotide sequence of the gene is shown as SEQ ID No. 2; the insect cell is Sf9 cell.

4. The triple inactivated vaccine for pigs according to claim 1, wherein the porcine seneca virus has the microorganism deposit number: CGMCC No. 18851.

5. The triple inactivated vaccine for pigs according to claim 1, wherein the H3N2 subtype swine influenza virus microorganism deposit number is: CGMCC No. 14740.

6. A method for preparing the bivalent inactivated vaccine for pigs of claim 1, comprising:

(1) amplifying the recombinant baculovirus expressing the O-type FMDV VP1 gene and then inactivating the recombinant baculovirus; amplifying the porcine Seneca virus and then inactivating the porcine Seneca virus; (2) uniformly mixing inactivated recombinant baculovirus solution expressing O-type FMDV VP1 gene, inactivated swine epinakai virus solution and inactivated H3N2 subtype swine influenza virus solution to obtain a water phase; (3) heating the immunologic adjuvant to obtain an oil phase; (4) adding the oil phase into the water phase, mixing, and emulsifying.

7. The method according to claim 6, wherein the inactivated FMDV-vp1 recombinant baculovirus solution and porcine Seneca virus solution are mixed in step (2) to make FMDV-vp1 protein content not less than 40 μ g and porcine Seneca virus not less than 10 μ g in each vaccine portion^8.0TCID₅₀Swine influenza virus of not less than 10^7.5EID₅₀。

8. The method of claim 6, wherein the immunoadjuvant is Montanide^TM206 adjuvant.

9. The method according to claim 6, wherein the immunoadjuvant is heated to 30 ℃ in step (3) to obtain an oil phase.

10. The method according to claim 5, wherein the volume ratio of the oil phase to the water phase in step (4) is controlled to be 1: 1; the emulsification comprises the following steps: firstly, adding the water phase into an emulsification tank, slowly stirring, then slowly adding the oil phase adjuvant, stirring for 30 minutes at 800r/min after adding, and standing for 30 minutes.