A kind of inactivated vaccine of E2 protein of swine fever virus and preparation method thereof

Abstract

This invention discloses an inactivated classical swine fever virus (CSF) E2 protein vaccine and its preparation method, belonging to the field of inactivated vaccine technology. The CSF E2 protein inactivated vaccine comprises an aqueous phase and an oil phase, wherein the aqueous phase includes sodium acetylsalicylate, CSF E2 protein, and PBS buffer solution. The preparation method includes the following steps: dissolving sodium acetylsalicylate and CSF E2 protein in PBS solution to obtain the aqueous phase; sterilizing the oil phase to obtain a sterilized oil phase; and adding the sterilized oil phase to the aqueous phase for emulsification to obtain the CSF E2 protein inactivated vaccine. The CSF E2 protein inactivated vaccine prepared by this invention improves vaccine safety while ensuring that it does not affect the antibody levels of the pig herd after vaccination, reduces the rise in body temperature after immunization, does not affect feed intake, and improves pig production performance 28 days after vaccination.

Description

Technical Field

[0001] The present invention relates to the technical field of inactivated vaccines, and in particular to an inactivated vaccine of E2 protein of swine fever virus and a preparation method thereof. Background Art

[0002] Classical swine fever is an acute, contact, septicemic infectious disease caused by the classical swine fever virus. The classical swine fever virus is widely prevalent worldwide. Clinically, it is mainly infected with atypical classical swine fever. Infected pigs show symptoms such as depression, fever, loss of appetite, and erythema or purple spots appear on the abdomen, armpits, ears, nose, limbs, etc. The infected pigs have a high morbidity and mortality rate, which has a serious impact on the development of the breeding industry and causes serious economic losses. There is no effective treatment for classical swine fever, and vaccination is the most effective way to control classical swine fever. In the 1950s, my country developed the world-famous attenuated rabbit vaccine for classical swine fever (strain C), and then developed a live classical swine fever vaccine cultured in primary cells and passaged cells. In the middle of the last century, my country proposed a strategy to eradicate classical swine fever, but it has not been purified yet. The reasons are complex and diverse. With the development of science and technology, the technology of using prokaryotic Escherichia coli or eukaryotic insect cells or genetically modifying cells to express and purify the main antigen E2 protein of classical swine fever has developed rapidly. The inactivated vaccine of the classical swine fever virus E2 protein has a good immune effect and can distinguish antibodies produced by wild virus infection and vaccine immunity. However, the use of inactivated vaccines is generally prone to safety issues, such as increased body temperature, decreased feed intake, vomiting and depression in pigs after immunization. In severe cases, it can even cause growth retardation and reduced weight gain rate. Summary of the invention

[0003] In order to solve the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide an inactivated vaccine of Classical Swine Fever Virus E2 protein and a preparation method thereof, so as to solve the problems of increased body temperature, decreased feed intake, and reduced weight gain of pigs after immunization with the existing inactivated vaccine.

[0004] The technical solution of the present invention to solve the above technical problems is as follows: an inactivated vaccine of classical swine fever virus E2 protein is provided, comprising an aqueous phase and an oil phase, wherein the aqueous phase comprises sodium acetylsalicylate, classical swine fever virus E2 protein and PBS buffer solution.

[0005] The beneficial effects of the present invention are as follows: the inactivated vaccine of the swine fever virus E2 protein provided by the present invention includes an aqueous phase and an oil phase, the aqueous phase provides antigen components and auxiliary components, stimulates the body to produce an immune response, and the oil phase acts as an adjuvant to enhance the immune effect of the vaccine and protect the stability of the antigen components. In the present invention, the swine fever virus E2 protein in the aqueous phase is a protective antigen, the purpose of which is to induce the pig body to produce an immune response against the swine fever virus; sodium acetylsalicylate has an antipyretic and analgesic effect, and sodium acetylsalicylate is added to the aqueous phase to ensure that the vaccine does not affect the antibody level of the pig herd after vaccination, thereby improving the safety of the vaccine, reducing the increase in body temperature of the pig herd after immunization, and not affecting feeding; and PBS is used to dilute and stabilize the antigen components to ensure the stability and activity of the vaccine during storage and use. For biologically active substances such as the swine fever virus E2 protein, the salt balance and pH buffering effect of PBS can maintain its most complete characteristics, thereby ensuring the effectiveness of the vaccine.

[0006] On the basis of the above technical solution, the present invention can also be improved as follows: Furthermore, the volume ratio of the water phase to the oil phase is 880-920:80-120; preferably, the volume ratio of the water phase to the oil phase is 900:100.

[0007] The beneficial effect of adopting the above further technical solution is that the vaccine can effectively play its role when the volume ratio of the water phase to the oil phase is 880-920:80-120, especially when the volume ratio of the water phase to the oil phase is 900:100, the effect is best.

[0008] Furthermore, the concentration of sodium acetylsalicylate in the aqueous phase is 0.02-0.04 g / ml; preferably, the concentration of sodium acetylsalicylate in the aqueous phase is 0.028 g / ml.

[0009] The beneficial effects of adopting the above-mentioned further technical scheme are as follows: the concentration of sodium acetylsalicylate in the aqueous phase will affect the immune response and the reaction after the response. When the concentration of sodium acetylsalicylate in the aqueous phase is 0.02-0.04g / ml, it can ensure that the safety of the vaccine is effectively improved without affecting the antibody level of the pig herd after vaccination, and the temperature increase of the pig herd after vaccination is reduced, and feeding is not affected. Especially when the concentration of sodium acetylsalicylate in the aqueous phase is 0.028g / ml, the effect is best.

[0010] Furthermore, the concentration of the classical swine fever virus E2 protein in the aqueous phase is 0.045-0.075 mg / ml; preferably, the concentration of the classical swine fever virus E2 protein in the aqueous phase is 0.056 mg / ml.

[0011] The beneficial effect of adopting the above-mentioned further technical scheme is that when the concentration of the classical swine fever virus E2 protein in the aqueous phase is 0.045-0.075 mg / ml, it can effectively induce the pig body to produce an immune response against the classical swine fever virus, especially when the concentration of the classical swine fever virus E2 protein in the aqueous phase is 0.056 mg / ml, the effect is best.

[0012] Furthermore, the oil phase is ISA 15VG.

[0013] The method for preparing the above-mentioned inactivated vaccine of swine fever virus E2 protein comprises the following steps: (1) Preparation of aqueous phase Sodium acetylsalicylate and classical swine fever virus E2 protein were dissolved in PBS solution to prepare an aqueous phase; (2) Preparation of oil phase sterilizing the oil phase to obtain a sterilized oil phase; (3) Emulsification The sterilized oil phase is added into the water phase for emulsification to prepare the inactivated vaccine of Classical Swine Fever Virus E2 protein.

[0014] Furthermore, the oil phase sterilization process is: the oil phase is sterilized by high pressure at 121° C. for 30 min.

[0015] Furthermore, the emulsification process is specifically as follows: adding the sterilized oil phase into the water phase, and emulsifying for 8-12 minutes at 20-30°C and 400-600r / min.

[0016] The present invention has the following beneficial effects: The inactivated swine fever virus E2 protein vaccine prepared by the present invention can improve the safety of the vaccine while ensuring that the antibody level of the pig herd is not affected after vaccination, reduce the increase in body temperature of the pig herd after vaccination, and not affect feeding, thereby improving the production performance of the pig herd 28 days after vaccination. DETAILED DESCRIPTION

[0017] The following examples are only used to explain the present invention and are not intended to limit the scope of the present invention. If no specific conditions are specified in the examples, the conditions are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0018] Embodiment 1: A method for preparing an inactivated vaccine of swine fever virus E2 protein comprises the following steps: (1) Preparation of PBS Weigh 8g NaCl, 0.2g KCl, 1.44g Na 2 HPO 4 and 0.24 g KH 2 PO 4Dissolve in 800 ml of deionized water, then dilute to 1 L with deionized water, sterilize at 121°C for 30 min, and cool for later use; (2) Preparation of aqueous phase Dissolve 25 g of sodium acetylsalicylate and 50 mg of classical swine fever virus E2 protein in 500 ml of PBS, and fill the volume to 900 ml with PBS; (3) Preparation of oil phase The ISA 15VG adjuvant was sterilized by high pressure sterilization at 121°C for 30 minutes and then cooled to obtain the sterilized ISA 15VG adjuvant; (4) Emulsification Take 100 ml of sterilized ISA 15VG adjuvant and slowly add it into the prepared aqueous phase under stirring, emulsify at 25°C and 600 r / min for 10 minutes, and store at 2-8°C after emulsification.

[0019] Embodiment 2: A method for preparing an inactivated vaccine of swine fever virus E2 protein comprises the following steps: (1) Preparation of PBS Weigh 8g NaCl, 0.2g KCl, 1.44g Na 2 HPO 4 and 0.24 g KH 2 PO 4 Dissolve in 800 ml of deionized water, then dilute to 1 L with deionized water, sterilize at 121°C for 30 min, and cool for later use; (2) Preparation of aqueous phase Dissolve 18 g of sodium acetylsalicylate and 40.5 mg of classical swine fever virus E2 protein in 500 ml of PBS, and fill the volume to 900 ml with PBS; (3) Preparation of oil phase The ISA 15VG adjuvant was sterilized by high pressure sterilization at 121°C for 30 minutes and then cooled to obtain the sterilized ISA 15VG adjuvant; (4) Emulsification Take 100 ml of sterilized ISA 15VG adjuvant and slowly add it into the prepared aqueous phase under stirring, emulsify at 25°C and 600 r / min for 10 minutes, and store at 2-8°C after emulsification.

[0020] Embodiment 3: A method for preparing an inactivated vaccine of swine fever virus E2 protein comprises the following steps: (1) Preparation of PBS Weigh 8g NaCl, 0.2g KCl, 1.44g Na 2 HPO 4and 0.24 g KH 2 PO 4 Dissolve in 800 ml of deionized water, then dilute to 1 L with deionized water, sterilize at 121°C for 30 min, and cool for later use; (2) Preparation of aqueous phase Dissolve 36 g of sodium acetylsalicylate and 67.5 mg of classical swine fever virus E2 protein in 500 ml of PBS, and fill the volume to 900 ml with PBS; (3) Preparation of oil phase The ISA 15VG adjuvant was sterilized by high pressure sterilization at 121°C for 30 minutes and then cooled to obtain the sterilized ISA 15VG adjuvant; (4) Emulsification Take 100 ml of sterilized ISA 15VG adjuvant and slowly add it into the prepared aqueous phase under stirring, emulsify at 25°C and 600 r / min for 10 minutes, and store at 2-8°C after emulsification.

[0021] Comparative Example 1: A method for preparing an inactivated vaccine of swine fever virus E2 protein comprises the following steps: (1) Preparation of PBS Weigh 8g NaCl, 0.2g KCl, 1.44g Na 2 HPO 4 and 0.24 g KH 2 PO 4 Dissolve in 800 ml of deionized water, then dilute to 1 L with deionized water, sterilize at 121°C for 30 min, and cool for later use; (2) Preparation of aqueous phase Dissolve 50 mg of classical swine fever virus E2 protein in 500 ml of PBS and fill the volume to 900 ml with PBS; (3) Preparation of oil phase The ISA 15VG adjuvant was sterilized by high pressure sterilization at 121°C for 30 minutes and then cooled to obtain the sterilized ISA 15VG adjuvant; (4) Emulsification Take 100 ml of sterilized ISA 15VG adjuvant and slowly add it into the prepared aqueous phase under stirring, emulsify at 25°C and 600 r / min for 10 minutes, and store at 2-8°C after emulsification.

[0022] Test example 1. Comparison of antibody levels Fifteen healthy susceptible piglets aged 28-35 days were divided into groups A1, B1 and C1, with 5 piglets in each group. Group A1 was injected with 2.0 ml / head of the inactivated vaccine of classical swine fever virus E2 protein prepared in Example 1, group B1 was injected with 2.0 ml / head of the inactivated vaccine of classical swine fever virus E2 protein without sodium acetylsalicylate prepared in Comparative Example 1, and group C1 was injected with 2.0 ml / head of PBS buffer. Second immunization was performed in the same route and dosage on the 21st day. Blood was collected and serum was separated on the 21st day after the second immunization, and the ELISA antibody titer of classical swine fever virus was detected. The geometric mean of the antibody titer of each group of piglets was calculated and compared and analyzed. The results are shown in Table 1.

[0023] Table 1 Comparison of antibody levels after immunization

[0024] As shown in Table 1, there is no significant difference in the antibody level after immunization between the inactivated classical swine fever virus E2 protein vaccine prepared by the present invention and the inactivated classical swine fever virus E2 protein vaccine without adding sodium acetylsalicylate, and both are higher than the PBS buffer group.

[0025] 2. Comparison of body temperature rise Fifteen healthy susceptible piglets aged 28-35 days were divided into groups A2, B2 and C2, with 5 pigs in each group. Group A2 was injected with 4.0 ml / head of the inactivated vaccine of classical swine fever virus E2 protein prepared in Example 1, group B2 was injected with 4.0 ml / head of the inactivated vaccine of classical swine fever virus E2 protein without adding sodium acetylsalicylate prepared in Comparative Example 1, and group C2 was injected with 4.0 ml / head of PBS buffer. The rectal temperature was measured once in the morning of 1-3 days before vaccination and 1-3 days after vaccination. The geometric mean of the increase in body temperature of each group of piglets was calculated and compared and analyzed. The results are shown in Table 2.

[0026] Table 2 Comparison of body temperature increase after immunization (unit: ℃)

[0027] As shown in Table 2, both the Classical Chinese Fever Virus E2 Protein Inactivated Vaccine Group and the Classical Chinese Fever Virus E2 Protein Inactivated Vaccine Group without Sodium Acetylsalicylate Added showed elevated body temperature after immunization, but the Classical Chinese Fever Virus E2 Protein Inactivated Vaccine Group was significantly lower than the Classical Chinese Fever Virus E2 Protein Inactivated Vaccine Group without Sodium Acetylsalicylate Added, and the phosphate buffer group showed almost no elevated body temperature. It can be seen that the addition of Sodium Acetylsalicylate to the inactivated vaccine can improve the safety of the vaccine and reduce the elevated body temperature of the pigs after immunization, while ensuring that the antibody level of the pigs is not affected after vaccination.

[0028] 3. Comparison of feeding situation Fifteen healthy susceptible piglets aged 28-35 days were divided into groups A2, B2 and C2, with 5 pigs in each group. Group A2 was injected with 4.0 ml / head of the inactivated vaccine of classical swine fever virus E2 protein prepared in Example 1, group B2 was injected with 4.0 ml / head of the inactivated vaccine of classical swine fever virus E2 protein without adding sodium acetylsalicylate prepared in Comparative Example 1, and group C2 was injected with 4.0 ml / head of PBS buffer. The feeding conditions of the piglets in each group after vaccination were observed and compared and analyzed. The results are shown in Table 3.

[0029] Table 3 Comparison of feeding situation after immunization

[0030] As shown in Table 3, the piglets in the Classical Chinese Fever Virus E2 Protein Inactivated Vaccine Group and the Phosphate Buffer Group ate actively and had normal appetite, and there was no decline in mental state that affected feeding. However, in the Classical Chinese Fever Virus E2 Protein Inactivated Vaccine Group without the addition of sodium acetylsalicylate, the piglets became depressed due to vaccination, resulting in reduced feed intake and appetite.

[0031] 4. Comparison of weight gain among pig groups Fifteen healthy susceptible piglets aged 28-35 days were divided into groups A2, B2 and C2, with 5 pigs in each group. Group A2 was injected with 4.0 ml / head of the inactivated vaccine of classical swine fever virus E2 protein prepared in Example 1, group B2 was injected with 4.0 ml / head of the inactivated vaccine of classical swine fever virus E2 protein without adding sodium acetylsalicylate prepared in Comparative Example 1, and group C2 was injected with 4.0 ml / head of PBS buffer. The piglets were weighed once on the day of vaccination and 28 days after vaccination. The geometric mean of the weight gain of each group of piglets was calculated and compared and analyzed. The results are shown in Table 4.

[0032] Table 4 Comparison of weight gain of pig groups after immunization (unit: kg)

[0033] As shown in Table 4, compared with the day of vaccination, the average weight gain of piglets in the inactivated classical swine fever virus E2 protein vaccine group and the phosphate buffer group were 12.8kg and 12.5kg, respectively, with no significant difference. The average weight gain of the inactivated classical swine fever virus E2 protein vaccine group without the addition of sodium acetylsalicylate was 10.0kg, which was significantly lower than that of piglets in the inactivated classical swine fever virus E2 protein vaccine group and the phosphate buffer group.

[0034] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. An inactivated vaccine of Classical Swine Fever Virus E2 protein, characterized in that: It comprises an aqueous phase and an oil phase, wherein the aqueous phase comprises sodium acetylsalicylate, classical swine fever virus E2 protein and PBS buffer solution.

2. The inactivated vaccine of classical swine fever virus E2 protein according to claim 1, characterized in that: The volume ratio of water phase to oil phase is 880-920:80-120.

3. The inactivated vaccine of classical swine fever virus E2 protein according to claim 2, characterized in that: The volume ratio of water phase to oil phase was 900:

100.

4. The inactivated vaccine of classical swine fever virus E2 protein according to any one of claims 1 to 3, characterized in that The concentration of sodium acetylsalicylate in the aqueous phase is 0.02-0.04 g / ml.

5. The inactivated vaccine of classical swine fever virus E2 protein according to claim 4, characterized in that: The concentration of sodium acetylsalicylate in the aqueous phase was 0.028 g / ml.

6. The inactivated vaccine of classical swine fever virus E2 protein according to any one of claims 1 to 3, characterized in that: The concentration of Classical Swine Fever Virus E2 protein in the aqueous phase is 0.045-0.075 mg / ml.

7. The inactivated vaccine of classical swine fever virus E2 protein according to claim 6, characterized in that: The concentration of Classical Swine Fever Virus E2 protein in the aqueous phase was 0.056 mg / ml.

8. The inactivated vaccine of classical swine fever virus E2 protein according to any one of claims 1 to 3, characterized in that: The oil phase is ISA15VG.

9. The method for preparing the inactivated vaccine of Classical Swine Fever Virus E2 protein according to any one of claims 1 to 8, characterized in that: The following steps are involved: (1) Preparation of aqueous phase Sodium acetylsalicylate and classical swine fever virus E2 protein were dissolved in PBS solution to prepare an aqueous phase; (2) Preparation of oil phase sterilizing the oil phase to obtain a sterilized oil phase; (3) Emulsification The sterilized oil phase is added into the water phase for emulsification to prepare the inactivated vaccine of Classical Swine Fever Virus E2 protein.

10. The preparation method according to claim 9, characterized in that: The specific emulsification process is: adding the sterilized oil phase into the water phase, and emulsifying for 8-12 minutes at 20-30°C and 400-600r / min.

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