Process method for producing attenuated porcine encephalitis B virus by optimizing BHK-21 cells

Abstract

The invention discloses a process method for producing an attenuated porcine encephalitis B virus by optimizing BHK-21 cells. The process method comprises a shake flask preparation process and a reactor preparation process, the shake flask preparation process is used for virus recovery; the reactor preparation process is used for virus amplification. Optimizing the technological process of preparing the suspension virus by shaking a flask, and optimizing parameters in the aspects of MOI value, cell density and freeze-thaw times after harvesting time; 2, optimizing the technological process of preparing the antigen by shaking a flask, and optimizing parameters in the aspects of MOI value, cell density, harvesting time and preservation conditions; and 3, optimizing the technological process of preparing the antigen by a reactor, wherein parameters in two aspects of pH and temperature are optimized. Compared with the prior art, the preparation method has the advantages that the porcine encephalitis B virus vaccine product can be efficiently and stably produced on an industrial scale through the optimization; powerful technical support is provided for production of porcine encephalitis B vaccines and diagnostic reagents, and the technical progress and industrial development in the field of porcine encephalitis B prevention and control are expected to be promoted.

Description

Technical Field

[0001] The invention belongs to the technical field of biopharmaceuticals, and particularly relates to a process for optimizing BHK-21 cells to produce attenuated porcine Japanese encephalitis virus. Background Art

[0002] Vaccination is a core method for the prevention and control of JE in pigs. High-quality JE antigens also play a key role in the development and production of JE diagnostic reagents. Therefore, the stable and efficient production of JE toxins and antigens is crucial for the prevention and control of JE.

[0003] Currently, the production of porcine JE toxin and antigens mostly relies on traditional processes, which present numerous drawbacks. Low cell growth efficiency in cell culture limits virus yield; imprecise inoculation methods affect infection efficiency, with existing processes often producing titers below 8.0 log10 TCID50 / ml. Virus harvesting and antigen preparation are complex processes, resulting in low recovery rates and inconsistent product quality. These issues not only increase production costs but also make it difficult to meet the growing market demand for porcine JE vaccines and diagnostic reagents. Therefore, the development of a novel, optimized process for the production of porcine JE toxin and antigens is urgent. Summary of the Invention

[0004] The purpose of this application is to provide a process for optimizing the production of attenuated porcine JE virus using BHK-21 cells. Through in-depth research into the biological characteristics of porcine JE virus, cell culture conditions, and viral infection mechanisms, and systematic optimization of multiple steps, from cell culture and virus inoculation to virus harvesting and antigen preparation, a highly efficient and stable process for producing porcine JE virus suspension toxin and antigen has been established. This process can significantly improve the yield and quality of porcine JE virus suspension toxin and antigen, reduce production costs, and enhance the controllability and stability of the production process.

[0005] A process for optimizing the production of attenuated porcine Japanese encephalitis virus by BHK-21 cells, comprising a shake flask preparation process and a reactor preparation process; The shake flask preparation process for virus recovery comprises the following steps: (1) Use serum-free medium TransVA-02 to revive BHK-21 cells and select cells from passages P5 to P30; (2) Resuspend the freeze-dried powder of the SA14-12-2 strain of porcine Japanese encephalitis virus in TransVA-02 medium to obtain a virus suspension; (3) The virus suspension was inoculated into cells with a density of 2×10 6 ~4×10 6BHK-21 cells at a concentration of 10 cells / ml were cultured in 5% CO2, 37°C, and 110 rpm, and the P1 virus seed was harvested within 24 to 72 hours. (4) The P1 virus strain was frozen and thawed at -80℃ for 0 to 3 times and then the titer was tested. The P1 virus strain with the determined titer was inoculated into a cell density of 2×10 6 ~4×10 6 cells / ml of BHK-21 cells, harvested the P2 virus seed, and stored at -80℃ and 2~8℃ respectively before testing the titer; The reactor preparation process for virus amplification includes the following steps: (1) inoculating stably passaged BHK-21 cells into a 7L BA reactor and culturing them in TransVA-02 medium. The reactor parameters are set to a controlled speed of 100 rpm and dissolved oxygen DO = 60%; (2) after the cell density doubles to 2.5×10 6 ~5×10 6 cells / ml, inoculate P1 virus, control the culture pH at 7.0-7.4, the virus amplification temperature at 33-36°C, and detect the titer after harvest.

[0006] Furthermore, in the shake flask preparation process, the MOI value of virus inoculation is 0.01-0.1, and the freeze-thaw method is a freeze-thaw cycle at -80°C and 32°C.

[0007] Furthermore, the seed passage density of the BHK-21 cells was 1×10 5 ~5×10 5 cells / ml, and TransVA-02 serum-free medium was used for both recovery and passaging.

[0008] Furthermore, the pH setting range is 7.0-7.4, the initial cell culture temperature is 37°C, and the temperature is adjusted to 33-36°C after virus inoculation.

[0009] Furthermore, the storage conditions of the P2 generation virus strain are: when stored at -80°C, the storage time is ≤30 days; when stored at 2~8°C, the storage time is ≤14 days.

[0010] Furthermore, the initial culture volume of the 7L BA reactor was 1500 ml, the final culture volume after virus inoculation was 3000 ml, and the basal aeration rate was 0.05 L / min.

[0011] Compared with the existing technology, the advantages of this application are: the production process of the live attenuated porcine Japanese encephalitis virus vaccine, the entire process is divided into three parts for optimization, the first is to optimize the process of preparing suspended poison in shake flasks, through the optimization of parameters such as MOI value, cell density, and number of freeze-thaw cycles after harvest time; the second is to optimize the process of preparing antigens in shake flasks, through the optimization of parameters such as MOI value, cell density, harvest time, and storage conditions; the third is to optimize the process of preparing antigens in reactors, through the optimization of parameters such as pH and temperature. Through the above optimization, porcine Japanese encephalitis virus vaccine products can be produced efficiently and stably on an industrial scale; it provides strong technical support for the production of porcine Japanese encephalitis vaccines and diagnostic reagents, and is expected to promote technological progress and industrial development in the field of porcine Japanese encephalitis prevention and control. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 This is the overall optimization process parameter diagram of the present invention. DETAILED DESCRIPTION

[0013] In order to make the purpose, technical solutions and advantages of this application more clear, the following further describes this application in detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain this application and are not intended to limit this application.

[0014] Example 1:10 6.7 TCID50 / ml inoculum reactor amplified antigen 1. Use serum-free TransVA-02 medium to recover BHK-21 cells, 1×10 6 cells / ml recovery density, and wait until the cells double to 5×10 6 cells / ml for passage, the passage density was 1×10 6 cells / ml, and were subcultured 5 times before use.

[0015] 2. Resuspend 1 vial of SA14-12-2 strain of porcine JE virus seed virus with a titer of log10 TCID50 = 6.7 in 1 ml of TransVA-02 medium.

[0016] 3. Inoculate 0.868 ml of the resuspended virus suspension (MOI = 0.05) into 30 ml of 2 × 10 6 cells / mlcells / ml of BHK-21 cells.

[0017] 4. Harvest the P1 virus 48 hours after infection and test its titer.

[0018] 5. The steps for testing titer are as follows: 1) For titer testing, adherent BHK-21 cells were plated in 96-well plates using 10% FBS DMEM medium. 2) Using serum-free DMEM medium as diluent, dilute in deep-well plate wells. Take 100 μl of harvested virus solution and add 900 μl of diluent to make a gradient dilution to 10-7; 3) Remove all medium from the 96-well plate and add 100 μl of each of 10-5, 10-6, and 10-7 to each 96-well plate. Inoculate 3 wells of each dilution. Finally, each well is supplemented with 10% FBS DMEM medium (final system: 200 μl). 4) After 7 days of incubation at 37°C, the lesions were removed and observed; The final suspension toxicity value was calculated using the TCID50 calculation formula and was log10 TCID50=9.0.

[0019] 6. Add 1 L of fresh TransVA-02 to the 7 L BA reactor and set the parameters to 110 rpm, dissolved oxygen D0 = 60%, pH = 7.4, and temperature 35 °C.

[0020] 7. Add 500 ml of BHK-21 cells containing 1.5×10 6 cells / ml of culture medium, and diluted to a final density of 5×10 5 cells / ml, and cultured for 40 hours until the cell density reached 5×10 6 cells / ml; 8. Take 0.643 ml (MOI = 0.05) of thawed P1 seed virus and add it to 1.5 L of fresh TransVA-02 and connect it to the reactor.

[0021] 9. 40 hours after inoculation, the P2 virus was harvested and the titer was tested. The final antigen titer was log10TCID50=9.16.

[0022] Example 2:10 7.0 TCID50 / ml inoculum reactor amplified antigen 1. Use serum-free TransVA-02 medium to recover BHK-21 cells, 1×10 6 cells / ml recovery density, and wait until the cells double to 5×10 6 cells / ml for passage, the passage density was 1×10 6 cells / ml, and were subcultured 5 times before use.

[0023] 2. Resuspend 1 vial of SA14-12-2 strain porcine Japanese encephalitis virus seed virus with a titer of log10TCID50=7.0 in 1 ml of TransVA-02 medium.

[0024] 3. Inoculate 0.435 ml of the resuspended virus suspension (MOI = 0.05) into 30 ml of 2 × 10 6 cells / ml of BHK-21 cells.

[0025] 4. Harvest the P1 virus 48 hours after infection and test its titer.

[0026] 5. The steps for testing titer are as follows: 1) For titer testing, adherent BHK-21 cells were plated in 96-well plates using 10% FBS DMEM medium. 2) Using serum-free DMEM medium as diluent, dilute in deep-well plate wells. Take 100 μl of harvested virus solution and add 900 μl of diluent to make a gradient dilution to 10-7; 3) Remove all medium from the 96-well plate and add 100 μl of each of 10-5, 10-6, and 10-7 to each 96-well plate. Inoculate 3 wells of each dilution. Finally, supplement each well with 10% FBS DMEM medium (final volume 200 μl). 4) After 7 days of incubation at 37°C, the lesions were removed and observed; The final suspension toxicity value calculated using the TCID50 calculation formula was log10 TCID50=8.9.

[0027] 6. Add 1 L of fresh TransVA-02 to the 7 L BA reactor and set the parameters to 110 rpm, dissolved oxygen D0 = 60%, pH = 7.4, and temperature 35 °C.

[0028] 7. Add 500 ml of BHK-21 cells containing 1.5×10 6 cells / ml of culture medium, and diluted to a final density of 5×10 5 cells / ml, and cultured for 40 hours until the cell density reached 5×10 6 cells / ml.

[0029] 8. Take 0.684 ml (MOI = 0.05) of thawed P1 seed virus and add it to 1.5 L of fresh TransVA-02 and inoculate it into the reactor.

[0030] 9. 40 hours after inoculation, the P2 virus was harvested and the titer was tested. The final antigen titer was log10 TCID50 = 9.

[0031] Example 3:10 7.4 TCID50 / ml inoculum reactor amplified antigen 1. Use serum-free TransVA-02 medium to recover BHK-21 cells, 1×10 6 cells / ml recovery density, and wait until the cells double to 5×10 6 cells / ml for passage, the passage density was 1×10 6 cells / ml, and were subcultured 5 times before use.

[0032] 2. Resuspend 1 vial of SA14-12-2 strain of porcine JE virus seed virus with a titer of log10 TCID50 = 7.4 in 1 ml of TransVA-02 medium.

[0033] 3. Inoculate 0.173 ml of the resuspended virus suspension (MOI = 0.05) into 30 ml of 2 × 10 6 cells / ml of BHK-21 cells.

[0034] 4. Harvest the P1 virus 48 hours after infection and test its titer.

[0035] 5. The steps for testing titer are as follows: 1) For titer testing, adherent BHK-21 cells were plated in 96-well plates using 10% FBS DMEM medium. 2) Using serum-free DMEM medium as diluent, dilute in deep-well plate wells. Take 100 μl of harvested virus solution and add 900 μl of diluent to make a gradient dilution to 10-7; 3) Remove all medium from the 96-well plate and add 100 μl of each of 10-5, 10-6, and 10-7 to each 96-well plate. Inoculate 3 wells of each dilution. Finally, each well is supplemented with 10% FBS DMEM medium (final system: 200 μl). 4) After 7 days of incubation at 37°C, the lesions were removed and observed; The final suspension toxicity value was calculated using the TCID50 calculation formula and was log10 TCID50=9.0.

[0036] 6. Add 1 L of fresh TransVA-02 to the 7 L BA reactor and set the parameters to 110 rpm, dissolved oxygen D0 = 60%, pH = 7.4, and temperature 35 °C.

[0037] 7. Add 500 ml of BHK-21 cells containing 1.5×10 6 cells / ml of culture medium, and diluted to a final density of 5×10 5 cells / ml, and cultured for 40 hours until the cell density reached 5×10 6 cells / ml.

[0038] 8. Take 0.643 ml (MOI = 0.05) of thawed P1 seed virus and add it to 1.5 L of fresh TransVA-02 and inoculate it into the reactor.

[0039] 9. 40 hours after inoculation, the P2 virus was harvested and the titer was tested. The final antigen titer was log10TCID50=9.

[0040]

[0041]

[0042] According to the above embodiments, Figure 1 As shown in Tables 1 and 2, by systematically optimizing the cell generation range, MOI value, culture temperature, freeze-thaw cycles, and reactor parameters, a set of efficient processes suitable for industrial production was established, which significantly improved the yield (titer increased to 8.4-9.16 log10TCID50 / ml) and stability of porcine JE virus toxin and antigen.

[0043] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, who makes equivalent replacements or changes based on the technical solution and inventive concept of the present invention, should be covered by the scope of protection of the present invention.

Claims

1. A process for optimizing BHK-21 cells to produce attenuated porcine Japanese encephalitis virus, characterized in that: Including shake flask preparation process and reactor preparation process; The shake flask preparation process for virus recovery comprises the following steps: (1) Use serum-free medium TransVA-02 to revive BHK-21 cells and select cells from passages P5 to P30; (2) Resuspend the freeze-dried powder of the SA14-12-2 strain of porcine Japanese encephalitis virus in TransVA-02 medium to obtain a virus suspension; (3) The virus suspension was inoculated into cells with a density of 2×10 6 ~4×10 6 BHK-21 cells at a concentration of 10 cells / ml were cultured in 5% CO2, 37°C, and 110 rpm, and the P1 virus seed was harvested within 24 to 72 hours. (4) The P1 virus strain was frozen and thawed at -80℃ for 0 to 3 times and then the titer was tested. The P1 virus strain with the determined titer was inoculated into a cell density of 2×10 6 ~4×10 6 cells / ml of BHK-21 cells, harvested the P2 virus seed, and stored at -80℃ and 2~8℃ respectively before testing the titer; The reactor preparation process for virus amplification includes the following steps: (1) inoculating stably passaged BHK-21 cells into a 7L BA reactor and culturing them in TransVA-02 medium. The reactor parameters are set to a controlled speed of 100 rpm and dissolved oxygen DO = 60%; (2) after the cell density doubles to 2.5×10 6 ~5×10 6 cells / ml, inoculate P1 virus, control the culture pH at 7.0-7.4, the virus amplification temperature at 33-36°C, and detect the titer after harvest.

2. The process for optimizing BHK-21 cells to produce attenuated porcine Japanese encephalitis virus according to claim 1, characterized in that: In the shake flask preparation process, the MOI value of virus inoculation is 0.01-0.1, and the freeze-thaw method is a freeze-thaw cycle at -80°C and 32°C.

3. The process for optimizing BHK-21 cells to produce attenuated porcine Japanese encephalitis virus according to claim 1, characterized in that: The seed passage density of the BHK-21 cells was 1×10 5 ~5×10 5 cells / ml, and TransVA-02 serum-free medium was used for both recovery and passaging.

4. The process for optimizing BHK-21 cells to produce attenuated porcine Japanese encephalitis virus according to claim 1, characterized in that: The pH setting range is 7.0-7.4, the initial cell culture temperature is 37° C., and the temperature is adjusted to 33-36° C. after virus inoculation.

5. The process for producing attenuated porcine Japanese encephalitis virus by optimizing BHK-21 cells according to claim 1 or 2, characterized in that: The storage conditions of the P2 generation virus strain are: when stored at -80°C, the storage time is ≤30 days; when stored at 2~8°C, the storage time is ≤14 days.

6. The process for optimizing BHK-21 cells to produce attenuated porcine Japanese encephalitis virus according to claim 1, characterized in that: In the shake flask preparation process, the shaker culture parameters are 5% CO2, 110 rpm, and constant temperature shaking at 37°C.

7. The process for optimizing BHK-21 cells for producing attenuated porcine Japanese encephalitis virus according to claim 1, characterized in that: The initial culture volume of the 7 L BA reactor was 1500 ml, the final culture volume after virus inoculation was 3000 ml, and the basal aeration rate was 0.05 L / min.

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