Culture method of high-yield rabies vaccine virus harvesting liquid

Abstract

The invention discloses a culture method of a high-yield rabies vaccine virus harvesting liquid, belongs to the technical field of vaccines, and solves the problems of low rabies vaccine yield, high cost and inconsistent product quality in the prior art. The method comprises the following steps: resuscitating human diploid working library cells by adopting a culture bottle, and amplifying step by step until the cells are subcultured to a cell factory; and inoculating the cells into a bioreactor for microcarrier culture, after the cells grow compactly, subculturing the cells into the bioreactor for flaky carrier culture, after the cells on the flaky carrier grow compactly, inoculating rabies virus, and then harvesting virus liquid. According to the method, on one hand, the defects of long passage time and large workload when cells are cultured by only using a culture bottle are avoided; on the other hand, the lamellar carrier is used for performing final cell culture and virus culture, so that the cells can be prevented from being influenced by stirring shearing force, the harvesting time of the viruses is prolonged, and the harvesting amount of the viruses is increased.

Description

Technical Field

[0001] The invention belongs to the technical field of vaccines, and in particular relates to a method for culturing a high-yield rabies vaccine virus harvest liquid. Background Art

[0002] Rabies is an acute infectious disease caused by the rabies virus. It is zoonotic and is common in carnivorous animals such as dogs, wolves, and cats. People are mostly infected by being bitten by sick animals. Rabies in my country is mainly spread by dogs. There is still a lack of effective treatment for rabies. The mortality rate of people suffering from rabies is nearly 100%. Patients generally die of respiratory or circulatory failure within 3 to 6 days. The main preventive measure at present is rabies vaccine.

[0003] Rabies vaccine is a vaccine with a long history. Currently, rabies vaccines are mainly divided into nerve tissue vaccine, avian embryo vaccine and cell culture vaccine. Among cell culture vaccines, human diploid cell rabies vaccine has good safety because it does not contain exogenous protein and exogenous DNA components, and does not have the potential tumorigenicity and carcinogenicity risks of animal-derived cells. It is recommended by WHO as the gold standard vaccine.

[0004] Since human diploid cell rabies vaccine is produced using human cells, it is highly safe, free of xenobiotic contamination, has a low incidence of adverse reactions, good immunogenicity, and long duration of immunity, and is suitable for the entire population. Therefore, human diploid cell rabies vaccine is still in great demand. However, due to low production, high cost, and no price advantage, its market use is at an absolute disadvantage compared to other animal cell culture vaccines.

[0005] In the prior art, the main factor restricting the low yield of human diploid cell rabies vaccine is the high difficulty of human diploid cell culture process. Human diploid cells grow slowly and have a limited number of passages, resulting in limited production capacity. And the human diploid cells currently used are MRC-5 cells. Although they have a long history of use, they have a high cell generation and are less sensitive to rabies vaccines, while other human diploid cells in China are only used internally. In addition, the current human diploid cell rabies vaccine culture process is to use culture bottles for cell recovery and passage, and then passage to multiple culture bottles and then to 15L bioreactors, and then mixed inoculation, passage to 100L bioreactors, and harvest after 6 days of culture. Under this process, the cell passage time is long, and more culture bottles are used, which is very labor-intensive; at the same time, the 100L reactor uses microcarrier culture, and the cells and culture fluid are fully contacted by the agitator. The shear force is more harmful to the cells, resulting in poor growth of the cells in the later stage, and the virus harvest can only last about 8 days.

[0006] Therefore, providing a human diploid cell rabies vaccine culture method that reduces the number of cell passages and workload and obtains a rabies virus harvest fluid with high yield and good quality has become an urgent problem to be solved by those skilled in the art. Summary of the invention

[0007] The object of the present invention is to provide a method for culturing a high-yield rabies vaccine virus harvest fluid, so as to solve the problems of low yield, high cost and inconsistent quality of human diploid cell rabies vaccine in the prior art.

[0008] To achieve the above purpose, the technical solution adopted by the present invention is as follows: The present invention discloses a method for culturing a high-yield rabies vaccine virus harvested liquid, comprising: reviving human diploid working bank cells in a culture bottle, amplifying them step by step, and then passage them to a cell factory; then inoculating them into a bioreactor for microcarrier culture, passage them to a bioreactor for sheet carrier culture after the cells on the microcarrier grow densely, inoculating rabies virus after the cells on the sheet carrier grow densely, and then harvesting the virus liquid.

[0009] In some embodiments of the present invention, rabies virus is inoculated by monolayer adsorption infection.

[0010] Some embodiments of the present invention include the following steps: S1. Cell recovery: Take the working bank cells, recover them in a culture bottle, and culture them in a 37±0.5℃, 5% carbon dioxide incubator; S2. Cell passaging: After the revived cells grow densely, they are gradually enlarged in culture bottles until they are passaged to cell factories; S3. Microcarrier preparation and cell passaging: After sterilizing the microcarrier and reactor, add cell growth medium for pre-culture; after the cells in the cell factory grow densely, pass the cells onto the microcarrier; S4. Preparation of sheet carriers and cell passaging: After pre-incubation, the sheet carriers are transferred to the bioreactor, sterilized, and pre-cultured with growth medium; after the cells on the microcarriers grow densely, they are passaged to the sheet carriers; S5. Virus inoculation: After the cells on the sheet carrier grow densely, inoculate the virus; S6. Virus harvesting: After virus inoculation and culture, the virus is washed with maintenance medium. After the maintenance medium is washed, the virus liquid is continuously harvested.

[0011] In some embodiments of the present invention, in step S2, the subculture ratio is 1:6-10, preferably 1:8.

[0012] In some embodiments of the present invention, the microcarriers include Cytodex Ⅰ microcarriers; the sheet carriers include disk carriers; Preferably, the bioreactor used in step S3 and step S4 is a stirred reactor with a volume of 3 to 50 L.

[0013] In some embodiments of the present invention, the pre-culture parameters of step S3 are dissolved oxygen: >20%, pH value: 6.8-7.8, rotation speed: 40-60 rpm, and temperature: 37.0±1°C.

[0014] In some embodiments of the present invention, the pre-culture parameters of step S4 are dissolved oxygen: >20%, pH value: 6.8-7.8, rotation speed: 80-120 rpm, and temperature: 32.0-38.0°C.

[0015] In some embodiments of the present invention, in step S5, after the cells on the sheet carrier grow densely, the virus is inoculated at an MOI of 0.01 to 0.1.

[0016] In some embodiments of the present invention, in step S6, the virus is inoculated and cultured for 3 days before being replaced with a maintenance solution, and the virus solution is continuously harvested 24±4 hours after the maintenance solution is replaced.

[0017] In some embodiments of the present invention, the longest time for continuously harvesting the virus fluid is 20 days.

[0018] Compared with the prior art, the present invention has the following beneficial effects: The present invention first adopts cell bottles and cell factories for passage, then uses microcarriers for cell amplification, and then digests the cells on the microcarriers and inoculates them onto paper carriers in a production-scale bioreactor, i.e., adopts a step-by-step amplification culture mode of cell bottles → cell factories → microcarriers → paper carriers. On the one hand, the method of the present invention avoids the defects of long passage time and heavy workload of simply using culture bottles to culture cells; on the other hand, using sheet carriers for the final cell culture and virus culture can effectively protect cells from the influence of stirring shear force, thereby increasing the virus harvest time and the virus harvest amount.

[0019] By adopting the method of the present invention, the cell proliferation speed is fast, within 40PDL, and the population doubling level is about 27h, which greatly shortens the time required for cell passage compared with the cells used in the existing process.

[0020] The cell generation when the virus of the present invention is inoculated is 38PDL, which is two generations earlier than the previous process, laying a foundation for subsequent process expansion.

[0021] By transferring cells from microcarriers to sheet carriers, the time and workload required for cell amplification are reduced, and the sheet carriers can effectively protect the cells, increasing the number of virus harvests (the existing process can only harvest for 8 days, but after the process improvement of this patent, the virus can be harvested for 20 days), thereby increasing the virus harvest.

[0022] The virus liquid produced by this process has high antigen content and virus titer and stable quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Attached Figure 1 This is a graph showing the results of investigating different culture carriers in Example 1 of the present invention; Attached Figure 2 This is a graph showing the results of investigating different culture methods in Example 2 of the present invention; Attached Figure 3 This is a comparison chart of the antigen content of Example 3 of the present invention and Example 1; Attached Figure 4 It is a comparison chart of the virus titer of Example 3 of the present invention and Comparative Example 1. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they 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.

[0025] The present invention discloses a method for culturing a high-yield rabies vaccine virus harvested liquid, the method comprising: The human diploid working bank cells are revived in culture bottles, amplified step by step, and then subcultured to a cell factory; then inoculated into a bioreactor for microcarrier culture, and after the cells on the microcarriers grow densely, subcultured into a bioreactor for sheet carrier culture, and after the cells on the sheet carriers grow densely, inoculated with rabies virus, and then the virus liquid is harvested; The working bank cells used in the embodiments of the present invention are KH-02 cells Homo sapiens., which are derived from the China Center for Type Culture Collection, with a preservation number of CCTCC NO: C2023325, and were deposited in the China Center for Type Culture Collection on November 1, 2023. The preservation address is: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0026] Preferably, the rabies virus is inoculated by single-layer adsorption infection.

[0027] The culture method of the present invention comprises the following steps: S1. Cell recovery: Take the working bank cells, recover them in a culture bottle, and culture them in a 37±0.5℃, 5% carbon dioxide incubator; S2. Cell passaging: After the revived cells grow densely, they are gradually enlarged in culture flasks until they are passaged to cell factories; the passaging ratio is 1:6~10, preferably 1:8.

[0028] S3. Microcarrier preparation and cell passaging: After sterilizing the microcarrier and reactor, add cell growth medium for pre-culture; after the cells in the cell factory grow densely, pass the cells onto the microcarrier; The microcarriers include Cytodex Ⅰ microcarriers; The pre-culture parameters of step S3 are dissolved oxygen: >20%, pH value: 6.8-7.8, rotation speed: 40-60 rpm, and temperature: 37.0±1°C.

[0029] S4. Preparation of sheet carriers and cell passaging: After pre-incubation, the sheet carriers are transferred to the bioreactor, sterilized, and pre-cultured with growth medium; after the cells on the microcarriers grow densely, they are passaged to the sheet carriers; The sheet-like carrier includes a disk carrier.

[0030] The pre-culture parameters of step S4 are dissolved oxygen: >20%, pH value: 6.8-7.8, rotation speed: 80-120 rpm, and temperature: 32.0-38.0°C.

[0031] Preferably, the bioreactor used in S3 and S4 is a stirred reactor with a volume of 3 to 50 L.

[0032] S5. Virus inoculation: After the cells on the sheet carrier grow densely, inoculate the virus at an MOI of 0.01 to 0.1; S6. Virus harvesting: After 3 days of virus inoculation and culture, the virus was replaced with maintenance medium. 24±4h after replacement of maintenance medium, the virus liquid was continuously harvested.

[0033] The longest time for continuous virus fluid harvesting is 20 days.

[0034] In one embodiment of the present invention, the working bank cell generation is 23PDL; the cell recovery generation is 24PDL.

[0035] In one embodiment of the present invention, the revived cells are gradually amplified and passaged in a culture flask to 29PDL, and then passaged to a cell factory; In one embodiment of the present invention, the cells are passaged to 32PDL in a cell factory and then cultured on microcarriers.

[0036] In one embodiment of the present invention, when the cells on the microcarriers are passaged to 35 PDL, they are further passaged to sheet carriers.

[0037] In one embodiment of the present invention, the cell passage number is 38PDL when the virus is inoculated.

[0038] The working cell bank cells described in the embodiments of the present invention are all 23PDL.

[0039] Example 1 In this example, microcarriers and paper carriers from different manufacturers were used to culture the virus, as follows: S1. Cell recovery: Take a working cell bank cell, melt it in a 37℃ water bath, transfer it to a T75 cell culture flask with a pipette, add 40ml of MEM growth medium, and culture it in a 37℃, 5% carbon dioxide incubator.

[0040] S2. Cell passaging: When the cells grow into a dense monolayer in the T75 cell flask, use digestion solution to terminate the cell digestion and then pass the cells to a T300 cell culture flask; repeat this operation at a passaging ratio of 1:8 until the cells are passaged to 3 cell factories.

[0041] S3. 3L bioreactor preparation and cell inoculation: After the microcarriers and sheet carriers from different manufacturers are treated according to the pre-treatment method recommended by the manufacturer, they are transferred into the 3L glass bioreactor. The 3L bioreactor is connected with the thread bottle, air filter, quick connector, etc. according to the experimental requirements, and the components are assembled, and the air tightness is checked (pressure maintenance value ≥ 0.1MPa), and high-pressure sterilization is performed; after the sterilization is completed, the cell growth medium is added for pre-culture; After the cells in the cell factory grow densely, they are digested and transferred to four 3L bioreactors; parameter settings: culture parameters are DO (dissolved oxygen): 50%, pH: 7.2, Motor (speed): 40-60rpm, T (temperature): 37.0℃.

[0042] S4. Cell medium replacement: After cell inoculation, replace half of the cell growth medium every 20 to 28 hours.

[0043] S5. Virus inoculation and culture: After the cells grow densely on the sheet carrier, inoculate the virus at 0.01-0.1 MOI, and the culture parameters are DO (dissolved oxygen): 50%, pH: 7.4, Motor (speed): 40-60rpm, T (temperature): 33.0℃. Replace the cell growth medium 44-52h after virus inoculation.

[0044] S6. Wash and replace the maintenance solution: On the third day after virus inoculation, wash and replace the maintenance solution three times.

[0045] S7. Virus harvesting: 20 to 28 hours after replacing the maintenance medium, start harvesting the virus solution continuously every day.

[0046] Antigen content and virus titer determination: ELISA method was used to determine the antigen content, and the results are attached. Figure 1 shown. Figure 1 The S in the horizontal axis represents the virus culture growth stage, in which the medium is changed once a day and is marked with numbers; H represents the virus harvest stage, in which the virus is harvested once a day and is marked with numbers.

[0047] The test results show that the antigen content of microcarriers is much lower than that of sheet carriers, indicating that sheet carriers are better than microcarriers in virus culture. By comparing the sheet carriers of three manufacturers, namely Jinyi Shengshi, Chu Kun and Saikecheng, it is found that the culture effects of sheet carriers from different manufacturers are different, and the sheet carrier culture effect of Chu Kun is the best.

[0048] The sheet carrier of Jinyi Shengshi in this embodiment refers to the sheet carrier provided by Zhejiang Jinyi Shengshi Bioengineering Co., Ltd.; The Chukun sheet carrier described in this embodiment refers to the sheet carrier provided by Shanghai Chukun Biotechnology Co., Ltd.; The sheet carrier of Saikecheng described in this embodiment refers to the sheet carrier provided by Wuhan Saikecheng Technology Co., Ltd.

[0049] Example 2 This example investigates the effects of monolayer adsorption infection culture and mixed culture on the antigen content of the harvested virus fluid, as follows: S1. Cell recovery: Take a working cell bank cell, melt it in a 37℃ water bath, transfer it to a T75 cell culture flask with a pipette, add 40ml of MEM growth medium, and culture it in a 37℃, 5% carbon dioxide incubator.

[0050] S2. Cell passaging: When the cells grow into a dense monolayer in the T75 cell flask, use digestion solution to terminate the cell digestion and then pass the cells to a T300 cell culture flask; repeat this operation at a passaging ratio of 1:8 until the cells are passaged to 3 cell factories.

[0051] S3. 3L bioreactor preparation and cell inoculation: After the sheet carrier is hydrated with PBS solution, it is transferred into a 3L glass bioreactor. The 3L bioreactor is connected with the thread bottle, air filter, quick connector, etc. according to the experimental requirements, and the components are assembled. The air tightness check (pressure maintenance value ≥ 0.1MPa) is carried out, and the high pressure sterilization is carried out; after the sterilization is completed, the cell growth medium is added for pre-culture; After the cells in the cell factory have grown densely, they are digested and passaged into two 3L bioreactors; One of the bioreactors was used as test group 1, using single-layer adsorption infection culture; the other bioreactor was used as test group 2, using mixed culture.

[0052] The culture parameters were DO (dissolved oxygen): 50%, pH: 7.2, Motor (rotation speed): 50 rpm, T (temperature): 37.0°C.

[0053] S4. Cell medium replacement: After cell inoculation, replace half of the cell growth medium every 20 to 28 hours.

[0054] S5. Virus inoculation and culture: After 7 days of cell culture in Experiment 1, the virus was inoculated at 0.01-0.1 MOI, and the culture parameters were DO (dissolved oxygen): 50%, pH: 7.4, Motor (speed): 40-60 rpm, T (temperature): 33.0℃. The cell growth medium was replaced 44-52 hours after virus inoculation and continued to be cultured; After the cells were digested in Experiment 2, the virus was inoculated into the cell suspension at 0.01-0.1 MOI, and adsorbed in a 37℃ water bath for half an hour, and shaken every half an hour. After adsorption, the cell virus suspension was inoculated into the bioreactor, and the culture parameters were set to DO (dissolved oxygen): 50%, pH: 7.2, Motor (speed): 40-60 rpm, T (temperature): 37.0℃ and continued to be cultured.

[0055] S6. Washing and replacing maintenance medium: In test group 1, the maintenance medium was washed and replaced three times 3 days after virus inoculation; in test group 2, the maintenance medium was washed and replaced after 7 days of culture.

[0056] S7. Virus harvesting: 20 to 28 hours after replacing the maintenance medium, start harvesting the virus liquid continuously every day.

[0057] Antigen content and virus titer determination: ELISA method was used to determine the antigen content, and the results are attached. Figure 2 shown. Figure 2 The S in the horizontal axis represents the virus culture stage, in which the medium is changed once a day and is marked with numbers; H represents the virus harvest stage, in which the virus is harvested once a day and is marked with numbers.

[0058] Analysis of results: The experimental results show that the antigen content of the virus liquid harvested by the single-layer adsorption infection culture method is higher than that by the mixed culture method, and its antigen content is still above 4IU / ml 24 days after harvest.

[0059] Example 3 This embodiment discloses a method for culturing a high-yield rabies vaccine virus harvest solution of the present invention, which is as follows: S1. Cell recovery: Take a working cell bank cell (23PDL), melt it in a 37℃ water bath, transfer it to a T75 cell culture flask with a pipette, add 40ml of MEM growth medium, and culture it in a 37℃, 5% carbon dioxide incubator.

[0060] S2. Cell passaging: When the cells grow into a dense monolayer in the T75 cell flask, use digestion solution to terminate the cell digestion and then pass the cells to a T300 cell culture flask; repeat this operation at a passaging ratio of 1:8 until the cells are passaged to 3 cell factories.

[0061] S3. 15L bioreactor preparation and cell inoculation: After hydrating the microcarrier (5g / L) with PBS solution, transfer it into the glass bioreactor; connect the bioreactor with the thread bottle, air filter, quick connector, etc. according to the experimental requirements, assemble all components, perform air tightness check (pressure maintenance value ≥ 0.1MPa), and sterilize under high pressure. After sterilization, add cell growth medium for pre-culture.

[0062] After the cells in the cell factory grow densely, they are digested and transferred to two 15L bioreactors; the culture parameters are DO (dissolved oxygen): 50%, pH: 7.2, Motor (speed): 40-60rpm, T (temperature): 37.0℃.

[0063] S4. 50L bioreactor preparation and cell inoculation: S41. Cleaning of BC-50L bioreactor: Soak the tank body with 0.5mol / L NaOH solution for 2h and drain it; then rinse and soak the reactor with injection water; wipe the outer surface of the tank body, control cabinet and support frame with 75% ethanol.

[0064] S42. Calibration of pH and DO electrodes: Calibrate the pH and DO electrodes and install them on the bioreactor.

[0065] S43. Reactor pipeline connection: After the bioreactor is cleaned, connect the pipelines and sampling bottles according to production requirements; confirm the sealing of the valve group.

[0066] S44. Processing of sheet carriers: Weigh 960g of sheet carriers, soak and activate the sheet carriers in 0.5mol / L NaOH solution for 30 minutes, rinse with injection water until neutral, and transfer to the cell culture tank. Clean, package and sterilize each valve group, install it in the original position of the fermentation tank, add 0.01mol / L 40L PBS (pH7.2) buffer solution into the tank body, confirm the sealing of the tank body, and sterilize the tank. After the high-pressure sterilization is completed, wait until the temperature drops to 37°C, drain the PBS buffer solution, and add 40L cell growth medium.

[0067] S45. Parameter control: Set and confirm pre-incubation parameters, DO (dissolved oxygen): 50%, pH: 7.2, Motor (speed): 80-120rpm, T (temperature): 37.0℃, ventilation (Air): automatic control, O 2 : Automatic control, CO2 : Automatic control.

[0068] S46. Cell inoculation: After the cells in the 15L bioreactor grow densely, drain all the culture medium, wash the cells with PBS cleaning solution, digest the cells with cell digestion solution, and when all the cells on the microcarriers are found to have fallen off by microscopic examination, add 4000mL of cell growth solution to terminate the digestion, then filter the cells through a cartridge filter and collect the cell suspension. After counting, inoculate the cell suspension into the 50L bioreactor.

[0069] S5. Cell medium replacement: After cell inoculation, replace half of the cell growth medium every 20 to 28 hours.

[0070] S6. Virus inoculation and culture: After the cells grow densely on the paper carrier, inoculate the virus at 0.01-0.1 MOI, and set the parameters to DO (dissolved oxygen): 50%, pH: 6.8-7.8, Motor (speed): 100rpm, T (temperature): 33.0±1℃. Replace the cell growth medium 44-52h after virus inoculation.

[0071] S7. Wash and replace maintenance solution: On the 4th day after virus inoculation, wash and replace the reactor with maintenance solution 3 times.

[0072] S8. Virus harvesting: 20-28 hours after the maintenance solution was replaced, the virus solution was harvested continuously, once a day. Three batches of samples were produced using the above method. The antigen content in the three batches of virus solution was determined by ELISA method, and the virus titer in the three batches of virus solution was determined by plaque method. The results are shown in the attached Figure 3 Shown and attached Figure 4 shown. Figure 3 and Figure 4 The H on the middle horizontal axis represents the virus harvest stage, which is harvested once a day and is identified by numbers.

[0073] The results showed that the virus titer curves of the three batches of pilot tests were highly reproducible, indicating that the process was stable and could be applied to production in the future, laying the foundation for expanding the rabies vaccine market.

[0074] Comparative Example 1 This comparative example is to produce rabies vaccine virus harvest liquid according to traditional technology, and the process flow is as follows: after cell recovery, cell passage, and two-stage microcarrier passage, the virus is amplified to 100L and then the harvest is started. 70% of the culture volume is harvested every day. After 8 days of harvest, the virus yield decreases significantly and the harvest is stopped. The conditions of cell recovery, cell passage, and microcarrier passage are the same as those in Example 3. The output per 100L is about 30,000 to 40,000. This comparative example process can achieve 20 days of harvesting per batch and 70% of the culture volume each time by optimizing the passage route and the virus production platform carrier. In the future, it also has great potential for industrial production and market significance.

[0075] The antigen content in the virus solution of this comparative example was determined by ELISA method, and the virus titer in the virus solution of the comparative example was determined by plaque assay. The results are shown in the attached Figure 3 Shown and attached Figure 4 shown.

[0076] By the attached Figure 3 and attached Figure 4 It can be seen that compared with the rabies vaccine virus harvest liquid produced by the traditional process, the method of the present invention can achieve continuous harvesting of virus harvest liquid for 20 days, which is much higher than the 8 days of the traditional process, and its antigen content is higher than the existing process and shows an upward trend.

[0077] The above description is only a preferred embodiment of the present invention, which is merely illustrative of the present invention and does not limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made by ordinary technicians in this field to the technical solution of the present invention should fall within the scope of protection determined by the claims of the present invention.

Claims

1. A method for culturing a high-yield rabies vaccine virus harvest solution, characterized in that: include: The human diploid working bank cells are revived in culture flasks and amplified step by step until they are passaged to a cell factory; they are then inoculated into a bioreactor for microcarrier culture, and after the cells on the microcarriers grow densely, they are passaged to a bioreactor for sheet carrier culture, and after the cells on the sheet carriers grow densely, they are inoculated with rabies virus and the virus fluid is harvested.

2. The method for culturing a high-yield rabies vaccine virus harvested liquid according to claim 1, characterized in that: Rabies virus was inoculated using a single-layer adsorption infection method.

3. The method for culturing a high-yield rabies vaccine virus harvested liquid according to claim 1 or 2, characterized in that: The following steps are involved: S1. Cell recovery: Take the working bank cells, recover them in a culture bottle, and culture them in a 37±0.5℃, 5% carbon dioxide incubator; S2. Cell passaging: After the revived cells grow densely, they are gradually enlarged in culture bottles until they are passaged to cell factories; S3. Microcarrier preparation and cell passaging: After sterilizing the microcarrier and bioreactor, add cell growth medium for pre-culture; after the cells in the cell factory grow densely, pass the cells onto the microcarrier; S4. Preparation of sheet carriers and cell passaging: After pre-incubation, the sheet carriers are transferred to the bioreactor, sterilized, and pre-cultured with growth medium; after the cells on the microcarriers grow densely, they are passaged to the sheet carriers; S5. Virus inoculation: After the cells on the sheet carrier grow densely, inoculate the virus; S6. Virus harvesting: After virus inoculation and culture, the virus is washed with maintenance medium. After the maintenance medium is washed, the virus liquid is continuously harvested.

4. The method for culturing a high-yield rabies vaccine virus harvested liquid according to claim 3, characterized in that: In step S2, the subculture ratio is 1:6-10.

5. The method for culturing a high-yield rabies vaccine virus harvest fluid according to claim 3, characterized in that: The microcarriers include Cytodex Ⅰ microcarriers; the sheet carriers include disk carriers; The bioreactor used in step S3 and step S4 is a stirred reactor with a volume of 3 to 50 L.

6. The method for culturing a high-yield rabies vaccine virus harvest fluid according to claim 3, characterized in that: The pre-culture parameters of step S3 are dissolved oxygen: >20%, pH value: 6.8-7.8, rotation speed: 40-60 rpm, and temperature: 37.0±1°C.

7. The method for culturing a high-yield rabies vaccine virus harvest fluid according to claim 3, characterized in that: The pre-culture parameters of step S4 are dissolved oxygen: >20%, pH value: 6.8-7.8, rotation speed: 80-120 rpm, and temperature: 32.0-38.0°C.

8. The method for culturing a high-yield rabies vaccine virus harvest fluid according to claim 3, characterized in that: In step S5, after the cells on the sheet carrier grow densely, the virus is inoculated at an MOI of 0.01 to 0.

1.

9. The method for culturing a high-yield rabies vaccine virus harvested liquid according to claim 3, characterized in that: In step S6, the virus is inoculated and cultured for 3 days and then replaced with maintenance solution. 24±4 hours after the replacement of the maintenance solution, the virus solution is continuously harvested.

10. The method for culturing a high-yield rabies vaccine virus harvest solution according to claim 8, characterized in that: The longest time for continuous virus fluid harvesting is 20 days.

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