Preparation method and application of a porcine seneca valley virus disease inactivated vaccine

Abstract

The application discloses a preparation method and application of a Seneca Valley Virus disease inactivated vaccine for pigs, and the vaccine is prepared by using the following steps: suspending culture of Seneca Valley Virus, chromatographic purification of an antigen, and preparation of the vaccine by using an optimized water adjuvant. The Seneca Valley Virus inactivated vaccine prepared by the method has good safety, can induce piglets to generate high-level neutralizing antibodies, and effectively protects a pig group from the attack of a Seneca Valley Virus virulent virus for pigs.

Description

Technical Field

[0001] This invention belongs to the field of veterinary biological products, specifically relating to a method for preparing an inactivated vaccine against porcine Seneca Valley virus disease and its application. Background Technology

[0002] SVV is a newly discovered acute pathogen that causes swine idiopathic vesicular disease (SIVD) and epidemic transient neonatal losses (ETNL). The disease has a high morbidity rate, rapid course, and rapid spread, with a relatively low mortality rate, but a high mortality rate in piglets. Clinically, it mainly manifests as vesicles and ulcers on the snout and coronal band of the hooves, lameness, lethargy, diarrhea in newborn piglets, and acute death. In 2015, the Seneca Valley outbreak was first reported in Guangdong Province, my country. In 2016 and 2017, the Seneca Valley outbreak continued to spread, breaking out in multiple provinces and cities across my country, including Guangdong, Fujian, Hunan, Henan, and Heilongjiang. Clinical symptoms are characterized by vesicular lesions, and occasionally, large numbers of young piglets die, causing huge economic losses to the pig industry and seriously threatening my country's public health security and the development of the livestock industry.

[0003] Currently, there are no available biological vaccines for the prevention and control of this disease. Control of porcine Seneca Valley disease relies on effective pig farm management. However, pig farming models in China are complex and diverse, and pig farm management is relatively backward. Therefore, the development of corresponding biological products to control the outbreak and spread of this disease is extremely urgent. Summary of the Invention

[0004] (a) Technical problems to be solved

[0005] The purpose of this invention is to address the shortcomings of existing Seneca Valley virus biological products by providing an effective and safe method for the large-scale preparation of an inactivated vaccine. The preparation method used for this inactivated vaccine is simple and safe, and the vaccine has good immunization effects.

[0006] (II) Technical Solution:

[0007] This invention is achieved through the following technical solution:

[0008] This invention provides a method for preparing an inactivated vaccine against porcine Seneca Valley virus disease, the specific steps of which are as follows:

[0009] 1) The specific preparation process of the porcine Seneca Valley virus antigen is as follows: Suspended BHK-21 cells are prepared using a bioreactor until the cell density reaches 3.0–5.0 × 10⁻⁶ cells / year. 6Cells / ml were inoculated with Seneca Valley virus at an MOI of 0.001–0.1. After inoculation, the bioreactor control parameters were set as follows: DO 30–60%, pH 7.0–7.4, stirring speed 80–100 rpm / min, and temperature 36.5–37.5℃. Samples were taken at regular intervals during the inoculation period, and cytopathic effects were observed by staining with 0.4% trypan blue. When the cell viability was ≤20%, the suspension cultured virus solution was harvested.

[0010] 2) Antigen Purification: After three freeze-thaw cycles, the harvested virus suspension was centrifuged at 8000 rpm for 30 min using continuous flow centrifugation. Cell debris was discarded, and the supernatant was collected. The supernatant was then filtered through a 500 kDa hollow fiber column three times to collect the crude virus solution. Further purification was performed using molecular sieve chromatography gel permeation to obtain the purified Seneca Valley virus solution.

[0011] 3) The specific inactivation process is as follows: Add 2% by volume of the sterile filtered BEI solution to the porcine Seneca Valley purified virus solution to make the final BEI concentration 0.08%. After thorough mixing, pour the solution into a container and start timing after the temperature rises to 30℃. Inactivate for 36 hours, stirring once every 120 minutes. After the timing is completed, add the filtered sterile 50% sodium thiosulfate solution to the inactivated virus solution to make the final concentration 2%. After thorough mixing, store the inactivated antigen at 2-8℃.

[0012] Vaccine preparation

[0013] The inactivated porcine Seneca Valley antigen is slowly added to the polymeric aqueous adjuvant, with the amount of polymeric aqueous adjuvant being 50% of the antigen mass. The stirring speed is controlled at 2000 rpm and the stirring temperature is below 25 degrees Celsius. After the antigen and adjuvant are evenly dispersed, the mixture is dispensed and labeled to obtain the porcine Seneca Valley aqueous adjuvant inactivated vaccine.

[0014] Preparation of adjuvants in this invention

[0015] The polymeric aqueous adjuvant is composed of the following components in weight percentages:

[0016]

[0017]

[0018] Preferably, the polymeric aqueous adjuvant consists of the following components in weight percentages:

[0019]

[0020] Weigh each component according to the above proportions, add them sequentially to a measured amount of injectable aqueous solution, control the stirring speed, mix, keep the temperature below 60℃, and after mixing evenly into a liquid, sterilize at 121℃ for 30 minutes, cool to room temperature, and obtain an aqueous adjuvant suitable for porcine Seneca Valley inactivated vaccine.

[0021] (III) Beneficial Effects

[0022] 1) The porcine Seneca Valley virus inactivated vaccine prepared in this invention employs a different method from previous vaccine preparation methods. The suspension culture method produces high antigen yield and good uniformity, saving labor costs. Optimization of the inoculation dosage, inoculation method, and reactor control parameters significantly increases the viral load and shortens the antigen harvesting time, thus reducing antigen production costs. The optimal inoculation dosage after optimization is an MOI of 0.01–0.05, the inoculation process is incubation at 37°C at low speed (60 rpm / min) for 2 hours, the virus harvesting time is approximately 24 hours, and the virus content is ≥10. 9.5 TCID 50 / ml.

[0023] 2) This invention also optimizes the composition of the adjuvant. The adjuvant of this invention is a polymeric aqueous adjuvant with good biocompatibility. Through extensive screening experiments, this invention has found that the combination of polyethylene glycol, propylene glycol, and polyglutamic acid has strong water solubility, biocompatibility, biodegradability, and non-toxicity, which can improve drug targeting, effectively enhance efficacy, and improve the performance of drugs or materials. Furthermore, experiments have shown that the combination of the three components produces a synergistic effect, which can significantly increase the level of immune antibodies against porcine Seneca Valley vaccine, while the vaccine exhibits good stability and safety.

[0024] 3) The inactivated vaccine of this invention has good safety and efficacy, and can induce animals to produce strong neutralizing antibodies after immunization. Since there are currently no vaccine-related biological products for this type of pathogen on the domestic or international market, this vaccine can effectively prevent the spread of SVV in pig herds, reduce the economic losses caused by the disease, and has broad application prospects. Detailed Implementation

[0025] Unless otherwise specified, the methods described in the following embodiments are conventional methods.

[0026] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0027] Example 1: Preparation of inactivated antigen of porcine Seneca Valley virus

[0028] I. Preparation of porcine Seneca Valley Virus Fluid

[0029] 1. BHK-21 suspension cell resuscitation

[0030] The frozen BHK-21 suspension cell line (BHK-21-BP-2, preservation number CGMCC NO.17588, cell line BHK-21-BP-2 can be found in CN110157659A) was rapidly thawed in a 37°C water bath. After thawing, fresh suspension culture medium was added to the cells at an initial density of 0.5–0.8 × 10⁶ cells / year. 6 Cells / ml were seeded in shake flasks, 2% newborn calf serum was added, and the flasks were incubated in a constant temperature shaker at 37°C and 120 rpm / min. Samples were taken periodically, stained with 0.4% trypan blue, and cell viability and cell doubling rate were observed under a microscope. Once the cell culture reached the normal doubling rate and cell viability exceeded 95%, the cells were passaged normally.

[0031] 2. Cell passage

[0032] Cell density reaches 3-5 × 10 6 When the cell count reaches 95% or higher, dilute and passage the cells using suspension culture medium to a final cell concentration of 0.5–0.8 × 10⁻⁶ cells / ml. 6 Cells / ml were cultured in a constant temperature shaker at 37℃ and 120 rpm / min to expand the cell seed.

[0033] 3. BHK-21 suspension cell culture scaled up stepwise in a bioreactor

[0034] 1) BHK-21 suspension cell culture in a 10L bioreactor

[0035] Cell seeding was expanded in shake flasks and then inoculated into a 10L bioreactor, with an initial cell density of 0.5–0.8 × 10⁶ cells / mL. 6 The reactor process parameters were set as follows: cells / ml, 36.5–37.5℃, pH 7.0–7.2, DO 30–60%, stirring speed 80–100 rpm. Cell viability and density were observed periodically. The cell density was increased to 3–5 × 10⁶ cells / ml. 6 When the cell count reaches 95% or higher, the culture is further expanded.

[0036] 2) Continuous scale-up culture of BHK-21 suspension cells in 10L-50L-100L bioreactors

[0037] BHK-21 suspension cells cultured in a 10L bioreactor were transferred to a 50L bioreactor, with an initial cell density of 0.5–0.8 × 10⁻⁶ cells / year. 6The reactor process parameters were set as follows: cells / ml, 36.5–37.5℃, pH 7.0–7.2, DO 30–60%, stirring speed 80–100 rpm. Cell viability and density were observed periodically. The cell density was increased to 3–5 × 10⁶ cells / ml. 6 When the cell count reaches 95% or higher, the culture is further expanded. Using the same culture process, the cells are scaled up to a 100L bioreactor for further culture.

[0038] 3) Production of porcine Seneca Valley virus strain in a 100L reactor

[0039] Using the 100L suspension culture cells prepared above, the virus suspension culture was inoculated and incubated with the virus until the porcine Seneca Valley virus was harvested:

[0040] When the cell density reaches 3-5×10 6 When the cell count / ml reached 95% or higher, Seneca Valley virus seed virus (SVV-ZM-201801, preservation number CGMCC NO.16396; Seneca Valley virus SVV-ZM-201801 can be found in CN109554352A) was inoculated with an MOI of 0.001–0.1. After incubation at 37℃ for 2 hours at 60 rpm / min, the reactor process parameters were set as follows: 36.5–37.5℃, pH 7.0–7.2, DO 30–60%, and stirring speed 80–100 rpm. Cell viability and cell density were observed periodically. When the cell viability was ≤20%, the virus solution was harvested, and the virus titer was measured. The virus content was ≥10. 9.5 TCID 50 / ml.

[0041] II. Antigen Purification

[0042] 1. Viral fluid and cell debris treatment

[0043] After the virus suspension culture was harvested and subjected to three repeated low-temperature freeze-thaw cycles, the virus solution was centrifuged at 8000 rpm for 30 min using a continuous flow centrifugation method. Cell debris precipitate was discarded and the supernatant was collected.

[0044] 2. Crude purification of virus solution

[0045] The viral supernatant was washed and filtered three times with 0.01M phosphate buffer through a 300-500KD hollow fiber column to collect the crude pure viral solution.

[0046] 3. Molecular sieve chromatography purification

[0047] The crude virus solution was purified by 4FF molecular sieve (GE) and then by molecular sieve chromatography gel purification. The first peak of the solution was collected, and collection was stopped when the absorbance of the absorbance curve dropped to the lowest point. The protein removal rate of the collected peak sample was determined using the Lowry Protein Assay Kit (Thermo). The purified Seneca Valley virus solution was then harvested.

[0048] III. Antigen Inactivation

[0049] 1. Add 2% by volume of the sterile filtered BEI solution to the porcine Seneca Valley virus solution to make the final BEI concentration 0.08%. After thorough mixing, pour the solution into a container and start timing after the temperature rises to 30°C. Inactivate the virus for 36 hours, stirring once every 120 minutes.

[0050] 2. After the timing is completed, add the filtered and sterilized 50% sodium thiosulfate solution to the virus inactivation solution to make the final concentration 2%. After thorough mixing, store the inactivated antigen at 2-8℃.

[0051] Example 2: Optimization of suspension culture conditions for porcine Seneca Valley virus

[0052] For the 100L virus culture process prepared above, key process parameters were further optimized, and the effects of inoculation process and inoculation dosage on virus production were compared:

[0053] 1. Optimization of virus reception process

[0054] Cells were cultured in suspension at 100L until the cell density reached 3–5 × 10⁻⁶. 6 When the cell count / ml reached a viability of over 95%, the virus was inoculated with Seneca Valley virus seed. Inoculation was performed using two methods: direct inoculation and incubation (37℃, 60 rpm / min, 2 h). The advantages and disadvantages of the two methods were compared. The results are shown in the table below:

[0055]

[0056] The experimental results show that optimizing the inoculation process can effectively shorten the virus harvesting time and produce a higher viral load.

[0057] 2. Optimization of drug delivery dosage

[0058] Cells were cultured in suspension at 100L until the cell density reached 3–5 × 10⁻⁶. 6 When the cell count / ml reached a viability of over 95%, the virus was inoculated with Seneca Valley virus seed virus. The antigen production effects of several batches with different inoculation doses (MOI) of seed virus (0.001, 0.01, 0.05, 0.1) were compared. The results are shown in the table below:

[0059] MOI (Minimum Intake) Time to harvest viral lesions (h) <![CDATA[Virus content (TCID 50 / ml)]]> 0.001 35 <![CDATA[10 9.0 ]]> 0.01 30 <![CDATA[10 9.8 ]]> 0.05 26 <![CDATA[10 10.5 ]]> 0.1 16 <![CDATA[10 9.6 ]]>

[0060] The experimental results show that the inoculum dosage has a significant impact on the virus harvesting time and viral load. When the inoculum dosage is controlled within the range of 0.01–0.05, the virus harvesting time can be shortened, and the viral load can be further increased to 10. 9.8 TCID 50 / ml or higher helps to shorten the time cost of standardized production and improve antigen titer.

[0061] Example 3: Preparation of an aqueous adjuvant for porcine Seneca Valley inactivated vaccine

[0062] Through extensive screening experiments, this invention has found that the combined use of polyethylene, propylene glycol, and polyglutamic acid can significantly improve the level of immune antibodies against porcine Seneca Valley vaccine, while the vaccine also exhibits good stability and safety.

[0063] To verify the effectiveness of the adjuvant of the present invention, the following adjuvant was prepared:

[0064] Polymer-water adjuvant combination 1:

[0065] According to the technical solution provided by this invention, the composition and mass percentage of the polymer-water adjuvant are as follows:

[0066] 82% aqueous solution for injection;

[0067] Polymer combination:

[0068]

[0069] Additives:

[0070] 2% caprylic / capric triglyceride;

[0071] 1,2-Propanediol 2%.

[0072] Polymer-water adjuvant combination 2:

[0073] According to the technical solution provided by this invention, the composition and mass percentage of the polymer-water adjuvant are as follows:

[0074] 84% aqueous solution for injection;

[0075] Polymer combination:

[0076]

[0077] Additives:

[0078] 2% caprylic / capric triglyceride;

[0079] 1,2-Propanediol 2%.

[0080] Polymer-water adjuvant combination 3:

[0081] According to the technical solution provided by this invention, the composition and mass percentage of the polymer-water adjuvant are as follows:

[0082] 84.8% aqueous solution for injection;

[0083] Polymer combination:

[0084]

[0085] Additives

[0086] 1% caprylic / decanoic triglyceride;

[0087] 1,2-Propanediol 5%;

[0088] Polymer-water adjuvant combination 4

[0089] According to the technical solution provided by this invention, the composition and mass percentage of the polymer-water adjuvant are as follows:

[0090] 81% aqueous solution for injection;

[0091] Polymer combination:

[0092]

[0093] Additives

[0094] 5% caprylic / decanoic triglyceride;

[0095] 1,2-Propanediol 1%;

[0096] Weigh out the polymer and adjuvant combination according to the ratio, add them sequentially to a quantitative amount of aqueous solution for injection, control the stirring speed, mix, keep the temperature below 60℃, mix evenly into a liquid, sterilize at 121℃ for 30 minutes, cool to room temperature, and obtain an aqueous adjuvant suitable for porcine Seneca Valley inactivated vaccine.

[0097] Comparative Example 1:

[0098] 84% aqueous solution for injection;

[0099] Polymer combination:

[0100] 3% beta-glucan;

[0101] Polypropylene glycol 600 6%;

[0102] Polyethylene glycol 8000 2%;

[0103] Additive: 3% caprylic / capric triglyceride;

[0104] 1,2-Propanediol 2%;

[0105] Comparative Example 2:

[0106] 90% aqueous solution for injection;

[0107] Polymer combination:

[0108] 3% beta-glucan;

[0109] Polyethylene glycol 8000 2%;

[0110] Additive: 3% caprylic / capric triglyceride;

[0111] 1,2-Propanediol 2%;

[0112] Comparative Example 3:

[0113] 86% aqueous solution for injection;

[0114] Polymer combination:

[0115] 3% beta-glucan;

[0116] Polypropylene glycol 600 6%;

[0117] Additive: 3% caprylic / capric triglyceride;

[0118] 1,2-Propanediol 2%;

[0119] Comparative Example 4

[0120] According to the technical solution provided by this invention, the composition and mass percentage of the polymer water adjuvant are as follows: 80% aqueous solution for injection;

[0121] Polymer combination:

[0122] 5% dextran;

[0123] Polypropylene glycol 600 8%;

[0124] Polyethylene glycol 8000 5%;

[0125] Additives

[0126] 1,2-Propanediol 2%;

[0127] Example 3: Vaccine Preparation

[0128] The inactivated porcine Seneca Valley virus antigen from Example 1 was slowly added to any of the polymeric aqueous adjuvant combinations 1-4 in Example 2, with the amount of polymeric aqueous adjuvant being 50% of the antigen mass. The stirring speed was controlled at 2000 rpm, and the stirring temperature was below 25 degrees Celsius. After the antigen and adjuvant were evenly dispersed, they were dispensed and labeled to obtain the corresponding porcine Seneca Valley aqueous adjuvant inactivated vaccines 1-4. Inactivated vaccines with adjuvants from Comparative Examples 1-4 were prepared using the same method to obtain Comparative Examples 1-4.

[0129] Example 4: Safety test of inactivated vaccine

[0130] The porcine Seneca Valley inactivated vaccines 1-4 prepared in Example 3 and the comparative vaccines 1-4 were used to screen 4-week-old piglets for antigen-antibody negativity. Ten antigen-antibody negative piglets from each group were selected for a safety evaluation test of the Seneca Valley virus inactivated vaccine. Grouping: As shown in Table 1, 10 piglets from each polymer water adjuvant combination 1-4 were randomly selected to receive an overdose (4ml / pig) of the inactivated vaccine 1-4; 10 piglets from each of the comparative vaccines 1-4 were randomly selected to receive an overdose (4ml / pig) of the comparative vaccine 1-4; and 2 piglets from the control group were not vaccinated and served as negative controls. Body temperature was measured daily from day 0 to day 7 of immunization; feed intake was observed for 14 consecutive days; and the presence of lumps at the injection site was observed and palpated.

[0131] Table 1. Body Temperature Monitoring After Inactivated Vaccine Immunization

[0132]

[0133]

[0134] The results are shown in Table 1. No significant increase in body temperature was observed in piglets immunized with the inactivated vaccine, and the pigs had normal feed intake and good mental state. The injection site was painless to the touch 5 days after immunization, and there were no lumps, indicating that the vaccine was safe for pigs.

[0135] Example 5: Monitoring of neutralizing antibody levels after immunization with inactivated vaccine

[0136] To evaluate the immunization efficacy of the inactivated vaccine, 43 pigs were selected for the immunization experiment. 25 immunized pigs were given inactivated vaccine groups 1-4 (groups 1-4) and control groups 1-4, with 2 ml of inactivated vaccine injected intramuscularly into the neck of each pig. 3 control pigs were not vaccinated and served as blank controls. A second immunization was given 21 days after the first immunization. Serum samples were collected at 0, 7, 14, 21, 28 and 35 days after immunization. The level of anti-porcine Seneca Valley virus antibody production in the serum was evaluated using a neutralization test.

[0137] The specific steps are as follows:

[0138] 1) All the swine serum to be tested was incubated in a 56℃ water bath for 30 minutes to inactivate it;

[0139] 2) Each serum sample was serially diluted 1:2, 1:4, 1:8, ... 1:2048 in a 96-well cell culture plate, with 4 replicates for each dilution. Each well contained 100 μL of diluted serum, and the SVV neutralizing antibody was used for assay.

[0140] 3) Dilute the virus solution to 200 TCID using cell maintenance medium. 50 Add 0.1 mL of the diluted virus solution to the serum diluted in the above steps, 100 μL per well, mix well, and incubate at 37°C in a 5% CO2 incubator for 1 h.

[0141] 4) BHK21 cells in good growth condition were passaged at a 1:3 ratio and added to 96-well plates at 100 μl / well. Positive and negative control groups were set up, including a positive control group with virus solution and one without. Cellular activity was incubated at 37℃ in a 5% CO2 incubator. Cellular pathogenesis was observed and recorded daily. Serum neutralizing antibody levels were calculated using the Reed-Muench method. The data were analyzed after measuring serum neutralizing antibody titers, and the results are shown in Table 2.

[0142] Table 2. Levels of neutralizing antibodies produced in animals after immunization with inactivated vaccines.

[0143]

[0144]

[0145] Table 2 shows that the content of polypropylene glycol in the adjuvant and the presence or absence of polyglutamic acid and decanoate affect the antibody levels produced in piglets after immunization with the inactivated vaccine. When the polypropylene glycol content was 6%, polymer-water adjuvant combination 2 showed the highest antibody levels. In the absence of polyglutamic acid and decanoate, antibody levels decreased (see comparative examples 1-4). However, in summary, vaccines 1-4 all achieved a protective antibody level of 1:64, with the highest antibody level reaching 1:2048. The control group, which was not vaccinated, showed no detectable neutralizing antibodies against porcine Seneca Valley Virus. This data provides a theoretical and practical basis for the clinical application of inactivated vaccines against porcine Seneca Valley Virus disease.

[0146] Example 6: Strong Poison Challenge Protection Test

[0147] Piglets were immunized with the inactivated vaccine prepared in Example 3. Fourteen days after the second immunization, the immunization dose was 2 ml. All pigs were challenged with the virulent Seneca Valley virus strain GD / 01. 5 ml of the vaccine was dripped into each nostril, 2.5 ml into each nostril. The results are shown in Table 3.

[0148] Table 3 Results of protection against potent poisoning

[0149] Group Quantity (heads) Number of cases Immunogroup 5 0 / 5 Challenge control group 3 3 / 3

[0150] As shown in Table 3, 3 / 3 of the challenge control group developed the disease, while none of the immune group developed the disease, demonstrating 100% protection. This proves that the porcine Seneca Valley virus inactivated vaccine of the present invention can resist the attack of virulent Seneca Valley virus.

[0151] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been presented with preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make certain modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the technical solution of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention, without departing from the content of the technical solution of the present invention, shall still fall within the scope of the technical solution of the present invention.

Claims

1. A method for preparing an inactivated vaccine against porcine Seneca Valley virus disease, comprising the following steps: 1) The specific preparation process of the porcine Seneca Valley virus antigen is as follows: Suspended BHK-21 cell lines were prepared using a bioreactor until the cell density reached 3.0~5.0 × 10⁻⁶ cells / year. 6 Cells / mL were inoculated with Seneca Valley virus at a dose of 0.001–0.1 MOI. After inoculation, the bioreactor control parameters were set as follows: DO 30–60%, pH 7.0–7.4, stirring speed 50–100 rpm / min, and temperature 36.5–37.5 °C. Samples were taken periodically, and cytopathic effects were observed using 0.4% trypan blue staining. When cell viability was ≤20%, the suspension cultured virus solution was harvested. The inoculation amount of the virus was 0.01–0.05 MOI, the temperature was 37 °C, the stirring speed was 60 rpm / min, and the incubation time was 2 h. 2) Antigen purification: After the above-harvested suspension culture virus solution was repeatedly frozen and thawed three times, it was centrifuged at 8000 rpm for 30 min using continuous flow centrifugation. Cell debris precipitate was discarded and the supernatant was collected. The supernatant was repeatedly washed and filtered through a 500 KD hollow fiber column three times to collect the crude pure virus solution. Then, it was purified by molecular sieve chromatography gel purification to obtain the purified Seneca Valley virus solution. 3) Virus inactivation: Add 2% by volume of the sterile filtered BEI solution to the porcine Seneca Valley purified virus solution to make the final BEI concentration 0.08%. After thorough mixing, pour the solution into a container and start timing when the temperature rises to 30℃. Inactivate for 36 hours, stirring once every 120 minutes. After timing, add the filtered sterile 50% sodium thiosulfate solution to the inactivated virus solution to make the final concentration 2%. After thorough mixing, store the inactivated antigen at 2-8℃. 4) Vaccine preparation Inactivated porcine Seneca Valley antigen was slowly added to a polymeric aqueous adjuvant, the amount of which was 50% of the antigen mass. The stirring speed was controlled at 2000 rpm, and the stirring temperature below 25 degrees Celsius. After the antigen and adjuvant were evenly dispersed, the mixture was dispensed and labeled to obtain the porcine Seneca Valley aqueous adjuvant inactivated vaccine. The suspended BHK-21 cell line was BHK-21-BP-2; the Seneca Valley virus strain was Seneca Valley virus strain SVV-ZM-201801. The polymeric aqueous adjuvant is composed of the following components in weight percentages: Glucan 3%~5%; Polypropylene glycol 600 3%~8%; Polyethylene glycol 8000 1%~2%; Polyglutamic acid 0.2%~2%; Caprylic / decanoic triglyceride 1%~5%; 1,2-Propanediol 1%~5%; The remainder is water for injection.

2. The method according to claim 1, characterized in that: The polymeric aqueous adjuvant is composed of the following components in weight percentages: 3% beta-glucan; Polypropylene glycol 600 6%; Polyethylene glycol 8000 2%; Polyglutamic acid 1%; Caprylic / decanoic triglyceride 2%; 1,2-Propanediol 2%; The remainder is water for injection.

3. The method according to claim 1, characterized in that: The preparation method of the polymeric aqueous adjuvant is as follows: Weigh each component according to the proportion of the polymer aqueous adjuvant components, add it to a quantitative amount of injectable aqueous solution, control the stirring speed, mix, keep the temperature below 60°C, mix evenly into a liquid, sterilize at 121°C for 30 minutes, cool to room temperature, and obtain an aqueous adjuvant suitable for porcine Seneca Valley inactivated vaccine.

4. The porcine Seneca Valley vaccine prepared by the method according to any one of claims 1-3.

5. A polymeric aqueous adjuvant suitable for porcine Seneca Valley vaccine, characterized in that: The polymeric aqueous adjuvant is made from the following components in weight percentages: Glucan 3%~5%; Polypropylene glycol 600 3%~8%; Polyethylene glycol 8000 1%~2%; Polyglutamic acid 0.2%~2%; Caprylic / decanoic triglyceride 1%~5%; 1,2-Propanediol 1%~5%; The remainder is water for injection.

6. The polymeric aqueous adjuvant according to claim 5, characterized in that: The polymeric aqueous adjuvant is made from the following components in weight percentages: 3% beta-glucan; Polypropylene glycol 600 6%; Polyethylene glycol 8000 2%; Polyglutamic acid 1%; Caprylic / decanoic triglyceride 2%; 1,2-Propanediol 2%; The remainder is water for injection.

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