CN110628731B - Preparation method of porcine circovirus type II-porcine Seneca valley virus bivalent inactivated vaccine antigen - Google Patents
Preparation method of porcine circovirus type II-porcine Seneca valley virus bivalent inactivated vaccine antigen Download PDFInfo
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Abstract
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 counting4BHK-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 ℃. According to the invention, two viruses of the porcine circovirus type II and the porcine Sernica valley are sequentially inoculated into the same cell for culture, the antigen harvesting time is determined according to the cytopathic degree, and the porcine circovirus type II-porcine Sernica valley virus bivalent inactivated vaccine antigen is produced, so that the production efficiency is improved, the production cost is saved, and the product quality is improved.
Description
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%.
The porcine circovirus type II in the step 2) is DBN-SX07 strain with the titer of 107.5TCID50/mL。
The porcine Seneca valley virus in the step 2) is SD02 strain with the titer of 109.4TCID50/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
Example 1
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:
BHK-21 cells cultured to a monolayer were digested with trypsin at a mass concentration of 0.25% by a conventional digestion method, then digested with MEM nutrient solution containing 8% fetal calf serum, counted, and diluted to a density of 2X 104BHK-21 cell suspension per mL;
2) inoculation of porcine circovirus type ii with porcine seneca valley virus:
porcine circovirus type II (DBN-SX 07 strain with titer of 107.5TCID50mL) was inoculated into the above BHK-21 cell suspension in an amount of 5% by volume of the cell suspension, cultured in a carbon dioxide incubator at 37 ℃ for 48 hours, and then the cell maintenance solution (MEM nutrient solution supplemented with 1% fetal bovine serum) was replaced and simultaneously inoculated with porcine Seikaga valley virus (SD 02 strain, titer 10)9.4TCID50mL), the inoculum size is 0.1% of the cell maintenance liquid, and then the cells are cultured in a carbon dioxide incubator at 37 ℃;
3) and (3) harvesting the antigen:
BHK-21 cells inoculated with two virus antigens are cultured in a carbon dioxide incubator at 37 ℃ for about 24 hours, the cells are harvested according to the cytopathic condition (harvested when the cytopathic condition reaches more than 80%), and virus liquid after the cells are harvested is repeatedly frozen and thawed for three times and then stored in an environment below 40 ℃.
The titers of the two viruses in the harvested virus fluid were determined as follows:
(1) and (3) determining the porcine circovirus type II titer:
refining with 5% MEM PK-15The virus solution was diluted 10-fold in cell suspension, and each dilution (10)-4 .0～10 -6 .0) The virus solution is added into a cell plate respectively, each dilution is 6 holes, each hole is 100 mu L, the temperature is 37 ℃, and the CO content is 5 percent2Culturing in incubator for 24 hr, changing 2% MEM cell maintaining solution, placing cell plate at 37 deg.C and 5% CO2Culturing in the incubator for 48h, fixing with 80% acetone for 0.5h, incubating the primary and secondary antibodies at 37 deg.C for one hour, and observing virus infection with fluorescence microscope. The porcine circovirus type II titer in the harvested virus suspension was determined to be 10 using Reed-Muench's calculation7.0TCID50/mL。
(2) Assay for the titer of seneca valley virus
BHK-21 cells were plated in 96-well plates with 5% CO at 37 deg.C2The culture box is used for culturing until the cell monolayer grows to 80 percent. The virus solution was diluted 10-fold with MEM cell maintenance medium containing 2% fetal bovine serum, and each dilution (10)-7 .0～10-9 .0) Respectively adding the virus solution into a cell plate, wherein each dilution is 6 holes, each hole is 100 mu L, each hole is additionally added with 100 mu L maintenance solution, and the cell plate is placed at 37 ℃ and 5% CO2The cells were incubated in the incubator for 4 days and observed for lesions and the cytopathic results recorded on day 4. The titer of the porcine seneca valley virus in the harvested virus suspension is 10 by using the Reed-Muench's calculation method9.0TCID50/mL。
Example 2
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:
BHK-21 cells cultured to a monolayer were digested with trypsin at a mass concentration of 0.23% by a conventional digestion method, then digested with MEM nutrient solution containing 8.5% fetal bovine serum, and the cells were counted and diluted to a density of 2X 104BHK-21 cell suspension per mL;
2) inoculation of porcine circovirus type ii with porcine seneca valley virus:
porcine circovirus type II (DBN-SX 07 strain with titer of 107.5TCID50/mL) was inoculated theretoIn the BHK-21 cell suspension, the inoculation amount is 5.5% of the volume of the cell suspension, the cell suspension is cultured in a carbon dioxide incubator at 37 ℃ for 48h, then the cell maintenance solution (MEM nutrient solution added with 1% fetal calf serum) is replaced, and simultaneously the porcine Seikaga valley virus (SD 02 strain with the titer of 10) is inoculated9.4TCID50mL), the inoculum size is 0.12% of the cell maintenance liquid, and then the cells are cultured in a carbon dioxide incubator at 37 ℃;
3) and (3) harvesting the antigen:
BHK-21 cells inoculated with two virus antigens are cultured in a carbon dioxide incubator at 37 ℃ for about 24 hours, the cells are harvested according to the cytopathic condition (harvested when the cytopathic condition reaches more than 80%), and virus liquid after the cells are harvested is repeatedly frozen and thawed for three times and then stored in an environment below 40 ℃.
The titers of the two viruses in the harvested virus fluid were determined as follows:
(1) and (3) determining the porcine circovirus type II titer:
the virus solution was diluted 10-fold with 5% MEM PK-15 cell suspension, and each dilution (10)-4 .0～10 -6 .0) The virus solution is added into a cell plate respectively, each dilution is 6 holes, each hole is 100 mu L, the temperature is 37 ℃, and the CO content is 5 percent2Culturing in incubator for 24 hr, changing 2% MEM cell maintaining solution, placing cell plate at 37 deg.C and 5% CO2Culturing in the incubator for 48h, fixing with 80% acetone for 0.5h, incubating the primary and secondary antibodies at 37 deg.C for one hour, and observing virus infection with fluorescence microscope. The porcine circovirus type II titer in the harvested virus suspension was determined to be 10 using Reed-Muench's calculation7.1TCID50/mL。
(2) Assay for the titer of seneca valley virus
BHK-21 cells were plated in 96-well plates with 5% CO at 37 deg.C2The culture box is used for culturing until the cell monolayer grows to 90 percent for standby. The virus solution was diluted 10-fold with MEM cell maintenance medium containing 2% fetal bovine serum, and each dilution (10)-7 .0～10-9 .0) Respectively adding the virus solution into a cell plate, wherein each dilution is 6 holes, each hole is 100 mu L, each hole is additionally added with 100 mu L maintenance solution, and the cell plate is placed at 37 ℃ and 5% CO2Cultured in an incubator for 4 days, andlesions were observed and cytopathic results recorded on day 4. The titer of the porcine seneca valley virus in the harvested virus suspension is 10 by using the Reed-Muench's calculation method9.05TCID50/mL。
Example 3
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:
BHK-21 cells cultured to a monolayer were digested with trypsin at a mass concentration of 0.27% by a conventional digestion method, then digested with MEM nutrient solution containing 8% fetal calf serum, counted, and diluted to a density of 2X 104BHK-21 cell suspension per mL;
2) inoculation of porcine circovirus type ii with porcine seneca valley virus:
porcine circovirus type II (DBN-SX 07 strain with titer of 107.5TCID50mL) was inoculated into the above BHK-21 cell suspension in an amount of 5.2% by volume of the cell suspension, cultured in a carbon dioxide incubator at 37 ℃ for 48 hours, and then the cell maintenance solution (MEM nutrient solution supplemented with 1% fetal bovine serum) was replaced and simultaneously inoculated with porcine Seikaga valley virus (SD 02 strain, titer 10)9.4TCID50mL), the inoculum size is 0.11% of the cell maintenance liquid, and then the cells are cultured in a carbon dioxide incubator at 37 ℃;
3) and (3) harvesting the antigen:
BHK-21 cells inoculated with the two virus antigens are cultured in a carbon dioxide incubator at 37 ℃ for about 24 hours, the cells are harvested according to the cytopathic condition (harvested when the cytopathic condition reaches 80%), and virus liquid after the cells are harvested is repeatedly frozen and thawed for three times and then stored in an environment below-40 ℃.
The titers of the two viruses in the harvested virus fluid were determined as follows:
(1) and (3) determining the porcine circovirus type II titer:
the virus solution was diluted 10-fold with 5% MEM PK-15 cell suspension, and each dilution (10)-4 .0～10 -6 .0) Respectively adding the virus solution into a cell plate, wherein each dilution is 6 holes, each hole is 100 mu L, and the temperature is 37℃,5％ CO2Culturing in incubator for 24 hr, changing 2% MEM cell maintaining solution, placing cell plate at 37 deg.C and 5% CO2Culturing in the incubator for 48h, fixing with 80% acetone for 0.5h, incubating the primary and secondary antibodies at 37 deg.C for one hour, and observing virus infection with fluorescence microscope. The porcine circovirus type II titer in the harvested virus suspension was determined to be 10 using Reed-Muench's calculation6.9TCID50/mL。
(2) Assay for the titer of seneca valley virus
BHK-21 cells were plated in 96-well plates with 5% CO at 37 deg.C2The culture box is used for culturing until the cell monolayer grows to 85 percent for standby. The virus solution was diluted 10-fold with MEM cell maintenance medium containing 2% fetal bovine serum, and each dilution (10)-7 .0～10-9 .0) Respectively adding the virus solution into a cell plate, wherein each dilution is 6 holes, each hole is 100 mu L, each hole is additionally added with 100 mu L maintenance solution, and the cell plate is placed at 37 ℃ and 5% CO2The cells were incubated in the incubator for 4 days and observed for lesions and the cytopathic results recorded on day 4. The titer of the porcine seneca valley virus in the harvested virus suspension is 10 by using the Reed-Muench's calculation method9.1TCID50/mL。
Claims (5)
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;
the cell maintenance liquid is MEM nutrient solution added with 1% fetal calf serum;
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 when the cytopathic effect reaches 80%, 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 107.5TCID50/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 109.4TCID50/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 3) storing the porcine circovirus type II-porcine Seneca valley virus bivalent inactivated vaccine antigen in an environment below-40 ℃.
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| CN103263666A (en) * | 2013-05-24 | 2013-08-28 | 北京大北农科技集团股份有限公司动物医学研究中心 | Duplex inactivated vaccine of porcine circovirus type 2 and porcine mycoplasma hyopneumoniae and preparation method of duplex inactivated vaccine |
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