Method for co-culturing porcine epidemic diarrhea virus and porcine rotavirus antigen
Through the co-culture of swine epidemic diarrhea virus and swine rotavirus antigen, the problems of long production time, large resource consumption and high cost of viral multivalent vaccines in the prior art have been solved, and the production efficiency, cost reduction and antigen quality have been improved.
Patent Information
- Application Number
- CN202411927750.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-25
- Publication Date
- 2025-03-21
AI Technical Summary
During the production process of existing viral multi-linked multivalent vaccines, multiple viral antigens need to be cultivated in different cells respectively, resulting in long production time, high resource consumption, high cost, and the complexity of animal diseases and mixed infection of multiple viruses increase economic losses in the farm and adverse reactions in animals.
The method of co-culture of swine epidemic diarrhea virus and swine rotavirus antigen is adopted. By subculturing the virus to a stable state in susceptible cells, and then co-culture of viruses in co-cultured cells, the virus co-existence state under the natural environment is simulated, and the yield and quality of viral antigens are improved.
This method greatly shortens the preparation time of viral antigen, improves production efficiency, reduces production cycle, reduces production costs, and improves the quality of antigens, supporting faster vaccine development processes.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of biological products, and specifically relates to a method for co-culturing porcine epidemic diarrhea virus and porcine rotavirus antigens. Background Art
[0002] Vaccines can prevent and control the occurrence and spread of infectious diseases. According to the different types of infectious diseases, vaccines can be divided into viral vaccines, bacterial vaccines and parasitic vaccines. Among them, infectious diseases caused by viral infection have received more attention and attention due to their variety, rapid spread, high morbidity and mortality rates. The development and use of viral vaccines, especially veterinary viral vaccines, are characterized by a wide variety and wide use.
[0003] For viral vaccines, currently, inactivated vaccines or live vaccines are mostly used for production. Therefore, culture substrates such as chicken embryos or cells must be used to culture viral antigens during the production process. Due to the disadvantages of using chicken embryos to prepare viral antigens, huge amounts of chicken embryos are used, a large amount of manpower is required for chicken embryo inoculation and harvesting operations during the production process, and the troublesome handling of chicken embryos after use, more and more viral antigens are prepared by cell culture. With the increase in the scale and centralization of animal farming, the occurrence and prevention and control of animal diseases have become more complicated. Multiple viruses coexist in the same farm, or mixed infections of multiple viruses occur, which brings huge economic losses to the farm. At the same time, since the same farm needs to be vaccinated with multiple viral vaccines, the vaccination of vaccines will cause stress and adverse reactions in animals, thereby reducing the feed conversion rate of animals and increasing the cost of farming. Therefore, the development and use of multi-linked and multi-valent vaccines have become an urgent need for farms, and more and more multi-linked and multi-valent vaccines have been developed and marketed.
[0004] At present, in the production process of viral multi-link and multivalent vaccines, each virus is usually cultured in different cells to obtain a single viral antigen, and finally multiple viral antigens are mixed in proportion to prepare a multi-link and multivalent vaccine. Since most production companies have limited production lines and produce a large number of products, it takes a long time to complete the production of multiple viral antigens. After the viral antigens are produced, they must be stored by refrigeration or freezing, which requires a large number of refrigeration or freezing areas and consumes a lot of electricity resources. How to achieve rapid production of multiple viral antigens, improve production efficiency, and reduce production costs is one of the problems that still need to be solved. Summary of the invention
[0005] The object of the present invention is to provide a method for co-culturing porcine epidemic diarrhea virus and porcine rotavirus antigens, so as to achieve the purpose of improving production efficiency, shortening the production cycle of multi-component and multivalent vaccines and reducing production costs.
[0006] The technical solution adopted to achieve the above purpose is to provide a method for co-culturing porcine epidemic diarrhea virus and porcine rotavirus antigen, comprising the following steps: (1) inoculating porcine epidemic diarrhea virus strain into porcine epidemic diarrhea virus strain susceptible cells and subculturing for 2 to 3 generations to obtain porcine epidemic diarrhea virus seed virus; (2) inoculating the porcine rotavirus strain into porcine rotavirus susceptible cells and subculturing them for 2 to 3 generations to obtain porcine rotavirus seed virus; (3) separately culturing porcine epidemic diarrhea virus susceptible cells and porcine rotavirus susceptible cells into monolayers, digesting, pipetting, diluting and co-culturing in sequence to obtain co-culture cell fluid; (4) culturing the co-cultured cells in the co-cultured cell solution to a monolayer and washing the cells to obtain a pre-cultured cell solution; (5) The porcine epidemic diarrhea virus seed virus and porcine rotavirus seed virus in step (1) and step (2) are diluted respectively and co-inoculated into the pre-cultured cell fluid for cultivation to obtain a virus culture fluid, and then the virus is collected to obtain a co-cultured virus antigen.
[0007] The beneficial effects of the above technical solution adopted in the present invention are as follows: the present invention first ensures the purity and activity of the virus by passage-culturing porcine epidemic diarrhea virus and porcine rotavirus in susceptible cells to a stable state (seed virus); then co-culturing the susceptible cells of the two viruses can simulate the coexistence of viruses in the natural environment, which is conducive to the reproduction of the virus and the expression of the antigen, thereby improving the yield and quality of the viral antigen; co-culturing the virus again can shorten the preparation time of the viral antigen; at the same time, the same volume of culture medium and maintenance liquid can prepare more viral antigens, avoiding the repeated labor and resource consumption required for culturing the two viruses separately, simplifying the preparation process and improving efficiency. In addition, the high-quality co-cultured viral antigens of the present invention are crucial for the development of vaccines against porcine epidemic diarrhea and porcine rotavirus infection. The antigens prepared by this method can be used for the preparation and evaluation of vaccines, which helps to accelerate the research and development process of new vaccines.
[0008] Preferably, the cells susceptible to porcine epidemic diarrhea virus strains are Vero cells; and the cells susceptible to porcine rotavirus are MA104 cells.
[0009] Preferably, step (3) comprises the following steps: separately culturing porcine epidemic diarrhea virus strain susceptible cells and porcine rotavirus susceptible cells to monolayer, digesting them with trypsin and beating them into single cells, and then diluting porcine epidemic diarrhea virus strain susceptible cells and porcine rotavirus susceptible cells to 0.5-2×10 5 / mL, the diluted porcine epidemic diarrhea virus strain susceptible cells and porcine rotavirus susceptible cells are evenly mixed in a volume ratio of 1~2:1~2 to obtain a cell mixture, and then co-culture is performed to obtain a co-culture cell solution.
[0010] More preferably, step (3) comprises the following steps: separately culturing porcine epidemic diarrhea virus strain susceptible cells and porcine rotavirus susceptible cells to monolayer, digesting them with trypsin and beating them into single cells, and then diluting porcine epidemic diarrhea virus strain susceptible cells and porcine rotavirus susceptible cells to 1×10 5 / mL, the diluted porcine epidemic diarrhea virus strain susceptible cells and porcine rotavirus susceptible cells were mixed evenly in a volume ratio of 1:1 to obtain a cell mixture, and then co-cultured to obtain a co-culture cell solution.
[0011] More preferably, the volume concentration of pancreatin is 0.03-0.07%; and the DMEM medium is a DMEM medium containing 8-12% calf serum by volume.
[0012] More preferably, the volume concentration of pancreatin is 0.05%; and the DMEM medium is a DMEM medium containing 10% calf serum by volume.
[0013] More preferably, the co-culture comprises the following steps: adding the cell mixture to a bioreactor containing 5-7 g / L of microcarriers, and then adding a culture medium 8-10 times the volume of the cell mixture for culturing, and controlling the culture conditions of the bioreactor to be: pH 7.2-7.4, stirring rate 60-80 r / min, time 46-50 h; the culture medium is a DMEM culture medium containing 8-12% calf serum by volume.
[0014] Preferably, the washing in step (4) comprises the following steps: washing the cells 2 to 3 times with PBS buffer, wherein the volume of PBS buffer used in each washing is the same as the volume of the co-cultured cell solution.
[0015] Preferably, the dilution in step (5) is performed using virus maintenance solution; the volume of the virus maintenance solution is the same as the volume of the pre-cultured cell solution; the porcine epidemic diarrhea virus seed virus and porcine rotavirus seed virus in step (1) and step (2) are diluted to a final concentration of 0.005-0.02 mol.
[0016] More preferably, the porcine epidemic diarrhea virus seed virus and porcine rotavirus seed virus in step (1) and step (2) are diluted to a final concentration of 0.01 mol.
[0017] More preferably, the virus maintenance solution is a DMEM culture medium containing trypsin at a mass concentration of 3-6 mg / L.
[0018] More preferably, the virus maintenance solution is a DMEM culture medium containing trypsin at a mass concentration of 5 mg / L.
[0019] Preferably, the culture conditions in step (5) are: pH 7.2-7.4, stirring rate 50-60 r / min, and time 46-50 h.
[0020] Preferably, the virus collection in step (5) comprises the following steps: firstly freezing and thawing the virus culture solution 3 to 4 times, then centrifuging at 5000 to 7000 r / min for 8 to 12 min and collecting the supernatant.
[0021] More preferably, the virus collection in step (5) comprises the following steps: firstly freezing and thawing the virus culture fluid three times, then centrifuging at 6000 r / min for 10 min and collecting the supernatant.
[0022] The present invention has the following beneficial effects: The method for co-culturing porcine epidemic diarrhea virus and porcine rotavirus antigens of the present invention can greatly shorten the preparation time of viral antigens; at the same time, the same volume of culture medium and maintenance fluid can prepare more viral antigens, thereby reducing the direct production cost of viral antigen preparation; in addition, due to the shortened production time and the reduced use of equipment, etc., indirect production costs such as labor costs, water, electricity and gas can be greatly reduced. DETAILED DESCRIPTION
[0023] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with embodiment.It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention, that is, the embodiments described are only part of the embodiments of the present invention, rather than all embodiments.If the specific conditions are not indicated in the embodiments, they are carried out according to the conditions recommended by the normal conditions or the manufacturers.If the manufacturer is not indicated in the reagents or instruments used, they are all conventional products that can be obtained by commercial purchase.
[0024] Therefore, the following detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making creative work are within the scope of protection of the present invention.
[0025] The features and performance of the present invention are further described in detail below in conjunction with embodiments.
[0026] Example 1 A method for co-culturing porcine epidemic diarrhea virus and porcine rotavirus antigens comprises the following steps: (1) inoculating a porcine epidemic diarrhea virus strain into Vero cells and subculturing them for three generations to obtain a porcine epidemic diarrhea virus seed virus; (2) inoculating the porcine rotavirus strain into MA104 cells and subculturing them for three generations to obtain porcine rotavirus seed virus; (3) After Vero cells and MA104 cells were cultured to monolayer, they were digested with 0.05% trypsin and blown into single cells. Vero cells and MA104 cells were diluted to 2×10 5 / mL, the diluted Vero cells and MA104 cells were mixed evenly at a volume ratio of 1:1 to obtain a cell mixture, and then the cell mixture was added to a bioreactor containing 6g / L microcarriers, and then 9 times the volume of the cell mixture was added with a DMEM medium containing 10% calf serum by volume for culture, and the culture conditions of the bioreactor were controlled to be: pH 7.2, stirring rate 60r / min, time 48h, to obtain a co-culture cell solution; (4) culturing the co-cultured cells in the co-cultured cell solution to a monolayer, washing the cells three times with PBS buffer, wherein the volume of PBS buffer used in each washing is the same as the volume of the co-cultured cell solution, to obtain a pre-cultured cell solution; (5) diluting the porcine epidemic diarrhea virus seed virus and porcine rotavirus seed virus in step (1) and step (2) to a final concentration of 0.005 mol using a DMEM medium containing 5 mg / L pancreatic enzyme, and then inoculating them into the pre-cultured cell solution for culture under the following conditions: pH 7.2, stirring rate 50 r / min, and culture time 48 h. During the culture period, sampling was performed every 6 h for observation and detection to obtain a virus culture solution; (6) The virus culture medium was repeatedly frozen and thawed three times, and then centrifuged at 6000 r / min for 10 min and the supernatant was collected to obtain the co-cultured virus antigen.
[0027] Example 2 A method for co-culturing porcine epidemic diarrhea virus and porcine rotavirus antigens comprises the following steps: (1) inoculating a porcine epidemic diarrhea virus strain into Vero cells and subculturing them for three generations to obtain a porcine epidemic diarrhea virus seed virus; (2) inoculating the porcine rotavirus strain into MA104 cells and subculturing them for three generations to obtain porcine rotavirus seed virus; (3) After Vero cells and MA104 cells were cultured to monolayer, they were digested with 0.03% trypsin and blown into single cells. Vero cells and MA104 cells were diluted to 0.5×10 5 / mL, the diluted Vero cells and MA104 cells were mixed evenly in a volume ratio of 1:1 to obtain a cell mixture, and then the cell mixture was added to a bioreactor containing 6g / L microcarriers, and then 9 times the volume of the cell mixture and DMEM medium containing 8% calf serum by volume was added for culture, and the culture conditions of the bioreactor were controlled as follows: pH 7.4, stirring rate 80r / min, time 46h, to obtain a co-culture cell solution; (4) culturing the co-cultured cells in the co-cultured cell solution to a monolayer, washing the cells three times with PBS buffer, wherein the volume of PBS buffer used in each washing is the same as the volume of the co-cultured cell solution, to obtain a pre-cultured cell solution; (5) diluting the porcine epidemic diarrhea virus seed virus and porcine rotavirus seed virus in step (1) and step (2) to a final concentration of 0.01 mol using a DMEM medium containing 3 mg / L pancreatic enzyme, and then inoculating them into the pre-cultured cell solution for culture under the following conditions: pH 7.4, stirring rate 60 r / min, time 46 h, sampling every 6 h during the culture period for observation and detection, to obtain a virus culture solution; (6) The virus culture medium was repeatedly frozen and thawed four times, and then centrifuged at 5000 r / min for 8 min and the supernatant was collected to obtain the co-cultured virus antigen.
[0028] Example 3 A method for co-culturing porcine epidemic diarrhea virus and porcine rotavirus antigens comprises the following steps: (1) inoculating a porcine epidemic diarrhea virus strain into Vero cells and subculturing them for three generations to obtain a porcine epidemic diarrhea virus seed virus; (2) inoculating the porcine rotavirus strain into MA104 cells and subculturing them for three generations to obtain porcine rotavirus seed virus; (3) After Vero cells and MA104 cells were cultured to monolayer, they were digested with 0.07% trypsin and blown into single cells. Vero cells and MA104 cells were diluted to 1×10 5 / mL, the diluted Vero cells and MA104 cells were mixed evenly in a volume ratio of 1:1 to obtain a cell mixture, and then the cell mixture was added to a bioreactor containing 6g / L microcarriers, and then a DMEM medium containing 12% calf serum by volume was added in a volume 10 times that of the cell mixture for culture, and the culture conditions of the bioreactor were controlled as follows: pH 7.2, stirring rate 60r / min, time 50h, to obtain a co-culture cell solution; (4) Cultivating the co-cultured cells in the co-cultured cell solution to a monolayer, washing the cells twice with PBS buffer, wherein the volume of PBS buffer used in each washing is the same as the volume of the co-cultured cell solution, to obtain a pre-cultured cell solution; (5) diluting the porcine epidemic diarrhea virus seed virus and porcine rotavirus seed virus in step (1) and step (2) to a final concentration of 0.02 mol using a DMEM medium containing 6 mg / L pancreatic enzyme, and then inoculating them into the pre-cultured cell solution for culture under the following conditions: pH 7.2, stirring rate 50 r / min, and culture time 50 h. During the culture period, sampling was performed every 6 h for observation and detection to obtain a virus culture solution. (6) The virus culture medium was repeatedly frozen and thawed four times, and then centrifuged at 7000 r / min for 12 min and the supernatant was collected to obtain the co-cultured virus antigen.
[0029] Comparative Example 1 A method for culturing porcine epidemic diarrhea virus comprises the following steps: (1) inoculating a porcine epidemic diarrhea virus strain into Vero cells and subculturing them for three generations to obtain a porcine epidemic diarrhea virus seed virus; (2) After Vero cells were cultured to a monolayer, they were digested with 0.05% trypsin and beaten into single cells. The Vero cells were then diluted to 1×10 using DMEM medium containing 10% calf serum. 5 / mL, adding the diluted Vero cells to a bioreactor containing 6g / L microcarriers, and then adding 9 times the volume of the diluted Vero cells and DMEM medium containing 10% calf serum by volume for culture, and controlling the culture conditions of the bioreactor to be: pH 7.2, stirring rate 60 / min, time for 48h, to obtain cultured cell fluid; (3) Cultivating the cells in the culture cell solution to a monolayer, washing the cells three times with PBS buffer, wherein the volume of PBS buffer used in each wash is the same as the volume of the culture cell solution, to obtain a pre-culture cell solution; (4) diluting the porcine epidemic diarrhea virus seed virus in step (1) to a final concentration of 0.01 mol using a DMEM medium containing 5 mg / L pancreatic enzyme, and then inoculating it into the pre-cultured cell solution for culture under the following conditions: pH 7.2, stirring rate 50 r / min, time 48 h, sampling every 6 h during the culture period for observation and detection, to obtain a virus culture solution; (5) The virus culture medium was repeatedly frozen and thawed three times, and then centrifuged at 6000 r / min for 10 min and the supernatant was collected to obtain porcine epidemic diarrhea virus antigen.
[0030] Comparative Example 2 A method for culturing porcine rotavirus antigen comprises the following steps: (1) inoculating porcine rotavirus strain into MA104 cells and subculturing for 3 generations to obtain porcine rotavirus seed virus; (2) MA104 cells were cultured to monolayers and digested with 0.05% trypsin and blown into single cells. MA104 cells were then diluted to 2×10 using DMEM medium containing 10% calf serum. 5 / mL, the diluted MA104 cells were added to a bioreactor containing 6g / L microcarriers, and then 9 times the volume of the diluted MA104 cells was added with DMEM medium containing 10% calf serum by volume for culture. The culture conditions of the bioreactor were controlled as follows: pH 7.2, stirring rate 60 / min, time 48h, and culture cell fluid was obtained; (3) Cultivating the cells in the culture cell solution to a monolayer, washing the cells three times with PBS buffer, wherein the volume of PBS buffer used in each wash is the same as the volume of the culture cell solution, to obtain a pre-culture cell solution; (4) diluting the porcine rotavirus seed virus from step (1) to a final concentration of 0.01 mol using a DMEM medium containing 5 mg / L pancreatin, and then inoculating it into the pre-cultured cell solution for culture under the following conditions: pH 7.2, stirring rate 50 r / min, and culture time 48 h. During the culture period, sampling was performed every 6 h for observation and detection to obtain a virus culture solution; (5) The virus culture medium was repeatedly frozen and thawed three times, and then centrifuged at 6000 r / min for 10 min and the supernatant was collected to obtain porcine rotavirus antigen.
[0031] Experimental example: Virus content detection 1. Virus content determination: The samples sampled at different time points in Example 1, Comparative Example 1 and Comparative Example 2 and the viral antigen finally harvested were serially diluted 10 times with DMEM containing 5 mg / L, and appropriate dilutions were inoculated into Vero cells and MA104 cells for virus content determination. Eight replicate wells were inoculated for each dilution, and the results were observed after culturing at 37° C., 5% CO2 for 72 to 96 h. The virus content was calculated according to the Reed-Muench method. The results are shown in Tables 1 and 2.
[0032] The results in Table 1 are the results of porcine epidemic diarrhea virus (PEDV) content determination. It can be seen from Table 1 that, compared with Comparative Example 1, the virus content of Example 1 is basically the same at 6h~24h after infection, but the virus content at 30h~36h after infection is slightly lower, but the difference is not significant.
[0033] Table 1 PEDV content determination results
[0034] The results in Table 2 are the results of PoRV (porcine rotavirus) content determination. It can be seen from Table 2 that, compared with Comparative Example 2, the virus content of Example 1 is basically the same at 6h~30h after infection, but the virus content at 36h after infection is slightly lower, but there is no significant difference.
[0035] Table 2 PoRV content determination results
[0036] 2. Fluorescence quantitative PCR detection of nucleic acid copy number: The samples sampled at different time points in Example 1, Comparative Example 1 and Comparative Example 2 and the co-cultured viral antigens finally harvested were subjected to fluorescence quantitative PCR detection using a fluorescence quantitative PCR method. At the same time, a plasmid containing the PEDV M gene and the PoRV NSP2 gene was used to establish a standard curve. The viral nucleic acid copy number was calculated according to the standard curve. The results are shown in Tables 3 and 4.
[0037] Table 3 shows the results of PEDV virus nucleic acid copy number detection. It can be seen from Table 3 that, compared with Comparative Example 1, the nucleic acid copy number of Example 1 is basically the same at 6h~24h after infection, and the viral nucleic acid copy number of Comparative Example 1 is slightly higher than that of Example 1 at 30h~36h after infection.
[0038] Table 3 PEDV nucleic acid copy number detection results
[0039] Table 4 is the detection results of PoRV virus nucleic acid copy number. It can be seen from Table 4 that, compared with Comparative Example 2, the nucleic acid copy number of Example 1 is basically the same at 6h~24h after infection, and the viral nucleic acid copy number of Comparative Example 2 is slightly higher than that of Example 1 at 30h~36h after infection.
[0040] Table 4 PoRV nucleic acid copy number detection results
[0041] From the results of virus content detection and virus nucleic acid copy number detection, the virus content and nucleic acid copy number of PEDV and PoRV in Example 1 and Comparative Examples 1 and 2 after infection are basically the same, but the virus content and nucleic acid copy number of PEDV and PoRV in Example 1 in the later stage of culture are lower than those in Comparative Examples 1 and 2. This may be due to the fact that in the later stage of virus antigen co-culture, as the nutrients in the cell system are consumed, the synthesis of the virus and other activities are affected. However, at 24 to 30 hours after infection, the content and nucleic acid copy number of PEDV and PoRV viruses both reach a peak, and the virus antigen co-culture obtained at this time has no significant difference from the single culture. In actual production, the preparation of virus antigens adopts the method of harvesting when the virus reaches the highest peak. Therefore, in actual operation, the two virus antigens prepared in Example 1 are not significantly different from those in Comparative Examples 1 and 2, and the benefits obtained in Example 1 far exceed those in Comparative Examples 1 and 2 at the same time and the same production cost, and have significant time and cost advantages.
[0042] The present invention has been described according to the above embodiments. It should be understood that the above embodiments do not limit the present invention in any form. Any technical solutions obtained by equivalent replacement or equivalent transformation shall fall within the scope of the present invention.
Claims
1. A method for co-culturing porcine epidemic diarrhea virus and porcine rotavirus antigen, characterized in that: The following steps are involved: (1) inoculating porcine epidemic diarrhea virus strain into porcine epidemic diarrhea virus strain susceptible cells and subculturing for 2 to 3 generations to obtain porcine epidemic diarrhea virus seed virus; (2) inoculating the porcine rotavirus strain into porcine rotavirus susceptible cells and subculturing them for 2 to 3 generations to obtain porcine rotavirus seed virus; (3) separately culturing porcine epidemic diarrhea virus susceptible cells and porcine rotavirus susceptible cells into monolayers, digesting, pipetting, diluting and co-culturing in sequence to obtain co-culture cell fluid; (4) culturing the co-cultured cells in the co-cultured cell solution to a monolayer and washing the cells to obtain a pre-cultured cell solution; (5) The porcine epidemic diarrhea virus seed virus and porcine rotavirus seed virus in step (1) and step (2) are diluted respectively and co-inoculated into the pre-cultured cell fluid for cultivation to obtain a virus culture fluid, and then the virus is collected to obtain a co-cultured virus antigen.
2. The method for co-culturing porcine epidemic diarrhea virus and porcine rotavirus antigen according to claim 1, characterized in that: The porcine epidemic diarrhea virus strain susceptible cells are Vero cells; the porcine rotavirus susceptible cells are MA104 cells.
3. The method for co-culturing porcine epidemic diarrhea virus and porcine rotavirus antigen according to claim 1, characterized in that: The step (3) comprises the following steps: taking porcine epidemic diarrhea virus strain susceptible cells and porcine rotavirus susceptible cells and culturing them into monolayers respectively, digesting them with trypsin and blowing them into single cells respectively, and then using DMEM culture medium to dilute the porcine epidemic diarrhea virus strain susceptible cells and porcine rotavirus susceptible cells to 0.5-2×10 5 / mL, the diluted porcine epidemic diarrhea virus strain susceptible cells and porcine rotavirus susceptible cells are evenly mixed in a volume ratio of 1~2:1~2 to obtain a cell mixture, and then co-culture is performed to obtain a co-culture cell solution.
4. The method for co-culturing porcine epidemic diarrhea virus and porcine rotavirus antigen according to claim 3, characterized in that: The volume concentration of the pancreatic enzyme is 0.03-0.07%; the DMEM culture medium is a DMEM culture medium containing 8-12% calf serum by volume.
5. The method for co-culturing porcine epidemic diarrhea virus and porcine rotavirus antigen according to claim 3, characterized in that: The co-culture comprises the following steps: adding a cell mixture into a bioreactor containing 5-7 g / L of microcarriers, and then adding a culture medium with a volume 8-10 times that of the cell mixture for culturing, wherein the culture conditions of the bioreactor are controlled as follows: pH 7.2-7.4, a stirring rate of 60-80 r / min, and a time of 46-50 h; and the culture medium is a DMEM culture medium containing 8-12% calf serum by volume.
6. The method for co-culturing porcine epidemic diarrhea virus and porcine rotavirus antigen according to claim 1, characterized in that: The washing in step (4) comprises the following steps: washing the cells 2 to 3 times with PBS buffer, wherein the volume of PBS buffer used in each washing is the same as the volume of the co-cultured cell solution.
7. The method for co-culturing porcine epidemic diarrhea virus and porcine rotavirus antigen according to claim 1, characterized in that: The dilution in step (5) is performed using virus maintenance solution; the volume of the virus maintenance solution is the same as the volume of the pre-cultured cell solution; the porcine epidemic diarrhea virus seed virus and porcine rotavirus seed virus in step (1) and step (2) are diluted to a final concentration of 0.005-0.02 mol.
8. The method for co-culturing porcine epidemic diarrhea virus and porcine rotavirus antigen according to claim 7, characterized in that: The virus maintenance solution is a DMEM culture medium containing 3-6 mg / L trypsin.
9. The method for co-culturing porcine epidemic diarrhea virus and porcine rotavirus antigen according to claim 1, characterized in that: The culture conditions in step (5) are: pH 7.2-7.4, stirring rate 50-60 r / min, and time 46-50 h.
10. The method for co-culturing porcine epidemic diarrhea virus and porcine rotavirus antigen according to claim 1, characterized in that: The virus collection in step (5) comprises the following steps: firstly freezing and thawing the virus culture solution 3 to 4 times, then centrifuging at 5000 to 7000 r / min for 8 to 12 minutes and collecting the supernatant.