阅读说明：本技术 猪圆环病毒ⅱ型-猪塞尼卡谷病毒二联灭活疫苗抗原的制备方法 (Preparation method of porcine circovirus type II-porcine Seneca valley virus bivalent inactivated vaccine antigen ) 是由 崔龙萍 江兴华 徐丽云 陈丽萍 包松英 于 2019-10-09 设计创作，主要内容包括：本发明公开了猪圆环病毒Ⅱ型-猪塞尼卡谷病毒二联灭活疫苗抗原的制备方法,1)将培养至单层的BHK-21细胞用胰酶消化,然后加入MEM营养液终止消化,细胞计数后稀释为密度为2×10<Sup>4</Sup>个/mL的BHK-21细胞悬液；2)将猪圆环病毒Ⅱ型接种BHK-21细胞悬液中,培养48h后更换细胞维持液并同时接种猪塞尼卡谷病毒,继续培养；3)接种两种病毒抗原的BHK-21细胞培养24h左右,根据细胞病变情况收获细胞,收获细胞后的病毒液反复冻融后置于-40℃以下环境中储存。本发明将猪圆环病毒Ⅱ型和猪塞尼卡谷两种病毒先后都接种于同一种细胞中进行培养,根据细胞病变程度决定抗原收获时间,生产猪圆环病毒Ⅱ型-猪塞尼卡谷病毒二联灭活苗抗原,提高了生产效率,节约了生产成本,提升了产品质量。(The invention discloses a preparation method of a porcine circovirus type II-porcine Seneca valley virus bivalent inactivated vaccine antigen, 1) a BHK-21 cell cultured to a single layer is digested by pancreatin, then MEM nutrient solution is added to stop the digestion, and the cell is diluted to the density of 2 multiplied by 10 after counting 4 BHK-21 cell suspension per mL; 2) inoculating porcine circovirus type II into BHK-21 cell suspension, culturing for 48h, changing cell maintenance liquid, inoculating porcine Saikaca valley virus, and culturing; 3) BHK-21 cells inoculated with two virus antigens are cultured for about 24h, the cells are harvested according to the cytopathic condition, and virus liquid after harvesting the cells is repeatedly frozen and thawed and then stored in an environment below 40 ℃. The invention inoculates two viruses of porcine circovirus type II and porcine seneca valley in sequence in the same cell for culture, determines the antigen harvesting time according to the cytopathic degree, and produces the porcine circovirus type II-porcine seneca valley virus IIThe combined inactivation vaccine antigen improves the production efficiency, saves the production cost and improves the product quality.)

1. The preparation method of the porcine circovirus type II-porcine Seneca valley virus bivalent inactivated vaccine antigen is characterized by comprising the following steps: which comprises the following steps:

1) preparation of BHK-21 cells:

digesting BHK-21 cells cultured to monolayer with pancreatin by conventional digestion method, adding MEM nutrient solution containing 8-8.5% fetal calf serum to stop digestion, counting cells, and diluting to density of 2 × 10⁴BHK-21 cell suspension per mL;

2) inoculation of porcine circovirus type ii with porcine seneca valley virus:

inoculating porcine circovirus type II into the BHK-21 cell suspension, wherein the inoculation amount is 5-5.5% of the volume of the cell suspension, culturing for 48h in a 37 ℃ carbon dioxide incubator, then replacing the cell maintenance liquid and inoculating porcine Seneca valley virus simultaneously, wherein the inoculation amount is 0.1-0.12% of the cell maintenance liquid, and then culturing in the 37 ℃ carbon dioxide incubator;

3) and (3) harvesting the antigen:

culturing BHK-21 cells inoculated with the two virus antigens in a carbon dioxide incubator at 37 ℃ for 22-26h, harvesting the cells according to the cytopathic condition, and repeatedly freezing and thawing the virus liquid after harvesting the cells to obtain the porcine circovirus type II-porcine Seneca valley virus bivalent inactivated vaccine antigen.

2. The method for preparing the porcine circovirus type II-porcine Seneca valley virus bivalent inactivated vaccine antigen according to claim 1, which is characterized in that: the mass concentration of the pancreatin in the step 1) is 0.23-0.27%.

3. The method for preparing the porcine circovirus type II-porcine Seneca valley virus bivalent inactivated vaccine antigen according to claim 1, which is characterized in that: the porcine circovirus type II in the step 2) is DBN-SX07 strain with the titer of 10^7.5TCID₅₀/mL。

4. The method for preparing the porcine circovirus type II-porcine Seneca valley virus bivalent inactivated vaccine antigen according to claim 1, which is characterized in that: the porcine Seneca valley virus in the step 2) is SD02 strain with the titer of 10^9.4TCID₅₀/mL。

5. The method for preparing the porcine circovirus type II-porcine Seneca valley virus bivalent inactivated vaccine antigen according to claim 1, which is characterized in that: and 2) adding 1% fetal calf serum into the cell maintenance solution to obtain the MEM nutrient solution.

6. The method for preparing the porcine circovirus type II-porcine Seneca valley virus bivalent inactivated vaccine antigen according to claim 1, which is characterized in that: and 3) harvesting the cells when the cytopathic effect reaches 80%.

7. The method for preparing the porcine circovirus type II-porcine Seneca valley virus bivalent inactivated vaccine antigen according to claim 1, which is characterized in that: and 4) storing the porcine circovirus type II-porcine Seneca valley virus bivalent inactivated vaccine antigen in an environment below-40 ℃.

Technical Field

The invention belongs to the field of biological products, and particularly relates to a preparation method of a porcine circovirus type II-porcine Seneca valley virus bivalent inactivated vaccine antigen.

Background

The porcine circovirus type II is a pathogenic antigen of the porcine circovirus disease, the clinical symptoms of the porcine circovirus disease are complex, the porcine circovirus disease is mainly manifested as postweaning multisystemic failure syndrome, porcine dermatitis and nephrotic syndrome, interstitial pneumonia, reproductive disorders, congenital tremor and the like, the porcine circovirus type II has high morbidity and mortality, and is one of the key prevention and control diseases of various large pig farms in China. The inoculation of the porcine circovirus type II vaccine can effectively prevent and control the disease, and is the most effective measure for preventing and controlling the disease in China at present.

The porcine seneca valley virus is also called porcine seneca virus, and is a small ribonucleic acid (RNA) virus which is discovered and classified in recent years and can cause porcine infection and morbidity. Clinically, the Seneca valley virus mainly causes vesicular lesions of pigs, which are very similar to the lesions of foot-and-mouth disease, vesicular stomatitis of pigs, vesicular eruption of pigs and the like, and obvious blisters appear in nasal rhynchopsis, hoof coronarium and the like of infected pigs, and are difficult to distinguish by naked eyes. The virus is an external pathogen, is introduced into China in 2015, and rapidly spreads in Fujian, Guangdong, Guangxi, Henan, Hebei, Shandong, Liaoning and other provinces in sequence, so that a large number of pig farms are attacked, the influence on newborn piglets is particularly large, and the fatality rate of the piglets can reach 30% -70%. So far, no commercial Sernica valley vaccine is available in China, once a pig farm is in an uncontrolled state, the prevention and control situation is severe, and the research and development of the virus vaccine are urgent.

The two viruses bring huge economic loss to the pig industry in China and seriously restrict the development of the pig industry in China. The research and development of the porcine circovirus type II-porcine Seneca valley virus bivalent inactivated vaccine have important significance. The traditional method for preparing the bivalent inactivated vaccine antigen is to respectively collect two virus antigens after two viruses are respectively propagated on respective sensitive cells, then to respectively inactivate the two antigens, and finally to mix the two inactivated antigens together for emulsification. The method has the following defects: 1. when the porcine circovirus type II is subjected to cell culture alone, cytopathic effect is not generated, which is not beneficial to determining the virus harvesting time. 2. The separate culture of the two antigens requires more manpower and material resources. 3. The phenomenon of uneven mixing of local antigens is easy to occur in the mixing and emulsification of the two antigens after the inactivation, and meanwhile, the process is complicated and low in efficiency.

Disclosure of Invention

The invention aims to provide a preparation method of a porcine circovirus type II-porcine Seikaga valley virus bivalent inactivated vaccine antigen, which changes the traditional preparation method of the bivalent inactivated vaccine antigen, improves the production efficiency, saves the production cost and improves the product quality.

In order to achieve the purpose, the invention adopts the following technical scheme:

the preparation method of the porcine circovirus type II-porcine Seneca valley virus bivalent inactivated vaccine antigen comprises the following steps:

1) preparation of BHK-21 cells:

digesting BHK-21 cells cultured to monolayer with pancreatin by conventional digestion method, adding MEM nutrient solution containing 8-8.5% fetal calf serum to stop digestion, counting cells, and diluting to density of 2 × 10⁴BHK-21 cell suspension per mL;

2) inoculation of porcine circovirus type ii with porcine seneca valley virus:

inoculating porcine circovirus type II into the BHK-21 cell suspension, wherein the inoculation amount is 5-5.5% of the volume of the cell suspension, culturing for 48h in a 37 ℃ carbon dioxide incubator, then replacing the cell maintenance liquid and inoculating porcine Seneca valley virus simultaneously, wherein the inoculation amount is 0.1-0.12% of the cell maintenance liquid, and then culturing in the 37 ℃ carbon dioxide incubator;

3) and (3) harvesting the antigen:

culturing BHK-21 cells inoculated with the two virus antigens in a carbon dioxide incubator at 37 ℃ for 22-26h, harvesting the cells according to the pathological condition of the cells, repeatedly freezing and thawing the virus liquid after harvesting the cells for three times to obtain the porcine circovirus type II-porcine Seneca valley virus bivalent inactivated vaccine antigen, and storing the porcine circovirus type II-porcine Seneca valley virus bivalent inactivated vaccine antigen in an environment at the temperature below 40 ℃.

The mass concentration of the pancreatin in the step 1) is 0.23-0.27%.

Step 2) the porcine circovirus type IIIs DBN-SX07 strain with the titer of 10^7.5TCID₅₀/mL。

The porcine Seneca valley virus in the step 2) is SD02 strain with the titer of 10^9.4TCID₅₀/mL。

And 2) adding 1% fetal calf serum into the cell maintenance solution to obtain the MEM nutrient solution.

And 3) harvesting the cells when the cytopathic effect reaches 80%.

The invention adopts the technical scheme that two viruses of porcine circovirus type II and porcine seneca valley are sequentially inoculated into the same cell for culture, the two viruses are simultaneously propagated, one cell culture solution is obtained, two virus antigen solutions are simultaneously obtained, the antigen harvesting time is determined according to the cytopathic degree, and the porcine circovirus type II-porcine seneca valley virus dual inactivated vaccine antigen is produced.

The invention has the advantages that;

1. the antigen harvesting time can be determined according to the cytopathic condition

The porcine circovirus type II does not produce cytopathic effect after infecting the cells, so the virus antigen is harvested in a fixed time harvesting mode in clinic, the BHK-21 cells are inoculated with the porcine circovirus type II and then inoculated with the porcine Seikaga valley virus, obvious cytopathic effect can be generated, and the virus antigen can be harvested according to the cytopathic effect.

2. Improve the working efficiency and save the production cost

The method can change the two antigens which are required to be separately cultured and harvested originally into simultaneous culture and harvest, inoculate two viruses in the same batch of cells in sequence for joint propagation and then harvest together, reduces the operation procedures, improves the working efficiency (5 days are required for culturing a batch of antigens originally, only 3 days are required by the invention), and can greatly reduce the use of cells, spinner flasks, serum and nutrient solution and save the production cost.

3. The two antigens are distributed more uniformly

In the traditional method, two antigens obtained by separate culture are mixed to prepare a bivalent vaccine, each antigen has cell fragments or cell aggregates containing virus particles, and the cell aggregates are often different in size, so that the virus content of the two antigens after mixing is uneven. The antigens cultured by the invention contain two virus diseases, and each virus-containing cell contains two virus antigens, so that manual mixing is not needed.

Detailed Description