Culture method for increasing goat pox virus content

Abstract

The invention discloses a culture method for increasing the content of goat pox viruses, which comprises the following steps: step 1, healthy cell culture: cryopreserved BHK cells are recovered and subcultured to a certain number, a basic culture medium is a DMEM culture medium containing 5% of newborn calf serum, and subculture is continued; 2, L-glutamine (0-4 mM) is added into the culture medium for continuous passage, and one bottle is taken and divided according to the proportion of 1: 8 during passage each time; 3, culturing the goat pox virus: adding 2mM of L-glutamine into the BHK cell growth solution, digesting, dispersing and passaging according to a conventional method, and propagating to 40 bottles of cells; 4, each group of virus maintenance liquid contains 2% of newborn calf serum and 2mM L-glutamine, and trehalose with different concentrations is added. According to the method, additives such as trehalose are added in the culture process, so that the infection ability of the virus to cells is improved, the replication efficiency of the virus is enhanced, and the infection and proliferation efficiency of the virus is improved by controlling the cell passage times and optimizing the culture medium.

Description

Technical Field

[0001] The invention relates to the technical field of goat pox virus, in particular to a culture method for increasing the content of goat pox virus. Background Art

[0002] Goat pox is an acute, febrile, contact infectious disease caused by goat pox virus. The disease can occur all year round and often breaks out in sensitive goats without immunity. Sick goats and recovered infected goats are the main sources of infection. The virus is often present in the pox fluid, pox scabs and secretions, and contaminates the surrounding environment, feed and drinking water with the shed scabs and secretions. The disease is transmitted through damaged mucous membranes and can also be indirectly transmitted through blood-sucking insects such as goat lice. The morbidity rate can reach 100%, and the mortality rate is generally not high, but the malignant goat pox can be as high as 50% or more. Lambs are most susceptible and have a high mortality rate. Humans can also be infected with goat pox.

[0003] At the beginning of the disease, the sheep's body temperature rises to 39.5-41.5℃, the spirit is depressed, and the conjunctiva is flushed; some stand still or lie on the ground, serous, mucous, and purulent secretions flow from the nostrils, and the breathing and pulse speed up; a few sheep have decreased appetite and reduced water intake, and most sheep stop eating and drinking. After 2-4 days, soybean or broad bean-sized erythema appears on the skin of the sick sheep, especially in the udder, lips, inner side of the limbs and other hairless or hairless areas, and develops into solid hard papules protruding from the surface of the skin; the papules of the lesions of some sheep merge with each other, making the skin uneven. If feeding management and symptomatic treatment are strengthened and there is no secondary infection, the body temperature of the sick sheep will gradually return to normal, appetite will gradually recover, and they will recover; if the body condition is poor, feeding management is poor or secondary infection is secondary, it may cause death.

[0004] Vaccine immunization is the best way to prevent goat pox. At present, goat pox vaccines are mainly attenuated live vaccines, which are made by inoculating goat pox virus into BHK cells, harvesting cell cultures, adding appropriate freeze-drying stabilizers, and freeze-drying in a vacuum. Summary of the invention

[0005] The purpose of this section is to summarize some aspects of embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the specification abstract and the invention title of this application to avoid blurring the purpose of this section, the specification abstract and the invention title, and such simplifications or omissions cannot be used to limit the scope of the present invention.

[0006] In view of the above problems existing in the existing culture method for increasing the content of goat pox virus, the present invention is proposed.

[0007] Therefore, the object of the present invention is to provide a culture method for increasing the content of goatpox virus, which effectively promotes the growth of BHK cells and increases the proliferation rate and yield of goatpox virus by optimizing key components such as L-glutamine concentration and trehalose concentration in the cell culture process.

[0008] In order to solve the above technical problems, the present invention provides the following technical solutions: a method for increasing the content of goat pox virus, comprising the following steps:

[0009] Step 1: Healthy cell culture: The frozen BHK cells were revived and subcultured. The basic culture medium was DMEM medium containing 5% newborn calf serum. The cells were divided into 5 groups, with 1 T75 square bottle in each group, and continued to be subcultured.

[0010] Step 2: Add L-glutamine (0-4 mM) to the culture medium for continued subculturing. Take one bottle and divide it into bottles at a ratio of 1:8 each time. Discard the remaining 7 bottles. When the cells grow into a dense monolayer, digest them with 0.25% trypsin solution and then divide them into bottles for culture. Each group was subcultured for 3 generations, and the time it took for each group of cells to grow into a dense monolayer was observed each time.

[0011] Step 3: Cultivation of goat pox virus: Add 2 mM L-glutamine to the BHK cell growth medium, digest and disperse the cells according to the conventional method, expand to 40 bottles of cells, divide them into 5 groups, inoculate goat pox virus, each group has 8 bottles, and add 25 ml of virus maintenance solution to each bottle;

[0012] Step 4: Each group of virus maintenance solution contains 2% newborn calf serum, 2mM L-glutamine, 2% seed virus, and different concentrations of trehalose;

[0013] Step 5: Discard the growth medium of BHK cells that have grown well, inoculate BHK cells with goatpox virus at 1% to 2% of the total amount of the maintenance medium, add cell maintenance medium, and culture at 37°C. Observe 1 to 2 times a day, harvest when the lesions reach more than 75%, and take samples to test the virus content;

[0014] Step 6: Dilute the goat pox virus solution in serum-free DMEM medium in 10-fold series, take 3 appropriate dilutions, inoculate 96-well cell plates, inoculate 5 wells of each dilution, 0.1 ml per well, add 0.1 ml of Vero cell suspension containing 200,000 to 300,000 cells per ml to each well, incubate at 37°C, 5% Culture in an incubator for 3 to 5 days, observe the cytopathic effect every day, and make a judgment on the 5th day. Calculate the TCID of goatpox virus based on the number of wells with cytopathic effect 50 .

[0015] As a preferred embodiment of the method for increasing the content of goatpox virus in the present invention, the goatpox virus is a weak strain cultured in goatpox testicular cells; and the virus content is determined using Vero cells.

[0016] As a preferred embodiment of the culture method for increasing the content of goat pox virus of the present invention, DMEM culture medium is used for both production and virus content determination, 5% newborn calf serum is added to the cell culture medium, and 2% newborn calf serum is added to the maintenance medium.

[0017] As a preferred embodiment of the culture method for increasing the content of goatpox virus of the present invention, the addition of L-glutamine in step 2 can accelerate the division of BHK cells and promote their growth. The addition of 2 mM L-glutamine has the best effect on BHK cells. When divided into bottles at a ratio of 1:8, BHK cells can grow into a dense monolayer in 44 hours, which is 22 hours faster than cells without glutamine.

[0018] As a preferred embodiment of the culture method for increasing the content of goat pox virus of the present invention, L-glutamine is prepared separately, stored in a -20°C refrigerator, added to the culture solution before use, and when the culture solution added with L-glutamine is stored in a 4°C refrigerator for more than 2 weeks, L-glutamine should be added again, the content of L-glutamine in the culture solution is 1-4 mM, 200 mM L-glutamine solution is prepared for storage, and added to the culture solution when used.

[0019] As a preferred embodiment of the method for increasing the content of goat pox virus in the present invention, the virus content in step 4 is 10 7.0 TCID 50 / ml.

[0020] As a preferred embodiment of the culture method for increasing the content of goatpox virus of the present invention, the addition of trehalose to the virus maintenance solution can significantly promote the proliferation of goatpox virus, accelerate its pathological process, and increase the content of goatpox virus. The best effect is achieved by adding 6-8 mg / ml of trehalose, and the pathological time is shortened by more than 16 hours compared with that without adding trehalose, and the virus content is increased by more than 1.21 titers.

[0021] As a preferred embodiment of the culture method for increasing the content of goatpox virus of the present invention, L-glutamine and trehalose are used for BHK cell culture and culture and proliferation of goatpox virus to promote and improve the yield and quality of goatpox vaccine.

[0022] Beneficial effects of the present invention:

[0023] 1. Improve virus yield: By optimizing the key components such as L-glutamine concentration and trehalose concentration in the cell culture process, the growth of BHK cells was effectively promoted, and the proliferation rate and yield of goat pox virus were increased.

[0024] 2. Optimize the cell culture environment: Using DMEM medium containing 5% newborn calf serum and an appropriate concentration of L-glutamine can provide a good growth environment for BHK cells, ensuring that the cells can reach the optimal state in a short time, thereby improving the culture efficiency of the virus.

[0025] 3. Improve the efficiency of virus infection: Adding additives such as trehalose during the culture process can help improve the virus's ability to infect cells and enhance the virus's replication efficiency. The addition of trehalose is particularly helpful in improving the stability and proliferation of the virus in BHK cells.

[0026] 4. Shorten the culture cycle: By controlling the number of cell passages and optimizing the culture medium, the time required for cell growth can be reduced, and the efficiency of virus infection and proliferation can be improved, thereby shortening the virus harvesting cycle.

[0027] 5. Improve the accuracy of virus quantitative detection: by inoculating Vero cells with 10-fold serial dilution of the virus liquid, the TCID of the virus can be accurately determined 50 , ensuring that the virus concentration during the virus culture process can meet the subsequent vaccine production and experimental needs. DETAILED DESCRIPTION

[0028] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0029] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0030] The present invention provides a culture method for increasing the content of goat pox virus, comprising the following steps:

[0031] Step 1: Healthy cell culture: The frozen BHK cells were revived and subcultured. The basic culture medium was DMEM medium containing 5% newborn calf serum. The cells were divided into 5 groups, with 1 T75 square bottle in each group, and continued to be subcultured.

[0032] Step 2: Add L-glutamine (0-4 mM) to the culture medium for continued subculturing. Take one bottle and divide it into bottles at a ratio of 1:8 each time. Discard the remaining 7 bottles. When the cells grow into a dense monolayer, digest them with 0.25% trypsin solution and then divide them into bottles for culture. Each group was subcultured for 3 generations, and the time it took for each group of cells to grow into a dense monolayer was observed each time.

[0033] Step 3: Cultivation of goat pox virus: Add 2 mM L-glutamine to the BHK cell growth medium, digest and disperse the cells according to the conventional method, expand to 40 bottles of cells, divide them into 5 groups, inoculate goat pox virus, each group has 8 bottles, and add 25 ml of virus maintenance solution to each bottle;

[0034] Step 4: Each group of virus maintenance solution contains 2% newborn calf serum, 2mM L-glutamine, 2% seed virus, and different concentrations of trehalose are added; the virus content of the seed virus is 10 7.0 TCID 50 / ml;

[0035] Step 5: Discard the growth medium of BHK cells that have grown well, inoculate BHK cells with goatpox virus at 1% to 2% of the total amount of the maintenance medium, add cell maintenance medium, and culture at 37°C. Observe 1 to 2 times a day, harvest when the lesions reach more than 75%, and take samples to test the virus content;

[0036] Step 6: Dilute the goat pox virus solution in serum-free DMEM medium in 10-fold series, take 3 appropriate dilutions, inoculate 96-well cell plates, inoculate 5 wells of each dilution, 0.1 ml per well, add 0.1 ml of Vero cell suspension containing 200,000 to 300,000 cells per ml to each well, incubate at 37°C, 5% Culture in an incubator for 3 to 5 days, observe the cytopathic effect every day, and make a judgment on the 5th day. Calculate the TCID of goatpox virus based on the number of wells with cytopathic effect 50 .

[0037] Among them, the goatpox virus strain is a weak strain cultured in goatpox testicular cells; BHK21 cells are used to prepare goatpox production virus and antigens, and Vero cells are used to determine the virus content; DMEM culture medium is used for both production and virus content determination, and 5% newborn calf serum is added to the cell culture medium and 2% newborn calf serum is added to the maintenance medium.

[0038] Among them, adding L-glutamine in step 2 can accelerate the division of BHK cells and promote their growth. Adding 2mM L-glutamine has the best effect on BHK cells. When divided into bottles at a ratio of 1:8, BHK cells can grow into a dense monolayer in 44 hours, which is 22 hours faster than cells without glutamine.

[0039] Furthermore, L-glutamine is prepared separately, stored in a -20°C refrigerator, and added to the culture medium before use. When the culture medium added with L-glutamine is stored in a 4°C refrigerator for more than 2 weeks, L-glutamine should be added again. The content of L-glutamine in the culture medium is 1-4mM. A 200mM L-glutamine solution is prepared for storage and added to the culture medium when used.

[0040] Furthermore, adding trehalose to the virus maintenance solution can significantly promote the proliferation of goatpox virus, accelerate its pathological process, and increase the content of goatpox virus. Adding 6-8 mg / ml of trehalose has the best effect. The pathological time is shortened by more than 16 hours compared with not adding trehalose, and the virus content is increased by more than 1.21 titers.

[0041] Specifically, L-glutamine and trehalose are used for BHK cell culture and goatpox virus culture and proliferation, promoting and improving the yield and quality of goatpox vaccine.

[0042] The examples are as follows:

[0043] 1. Healthy cell culture: The frozen BHK cells were revived and subcultured to a certain number. The basic culture medium was DMEM culture medium containing 5% newborn calf serum. The cells were divided into 5 groups, with 1 T75 square bottle in each group, and continued to be subcultured. A certain concentration of L-glutamine (0-4mM) was added to the culture medium for continued subculture. One bottle was taken and divided into bottles at a ratio of 1:8 each time (the remaining 7 bottles were discarded). When the cells grew into a dense monolayer, they were digested with 0.25% trypsin solution and then cultured in bottles. Each group was subcultured for 3 generations, and the time it took for each group of cells to grow into a dense monolayer was observed each time, as shown in Table 1:

[0044] Table 1 Effects of different concentrations of glutamine on Vero cells

[0045] 2. Goat pox virus culture: Add 2mM L-glutamine to the BHK cell growth medium, digest and disperse according to the conventional method, expand to 40 bottles of cells, divide into 5 groups and inoculate goat pox virus, each group has 8 bottles, and add 25ml virus maintenance liquid to each bottle. Each group of virus maintenance liquid contains 2% newborn calf serum, 2mM L-glutamine, 2% seed virus (virus content is 10 7.0 TCID 50 / ml), and different concentrations of trehalose were added, see Table 2 for details:

[0046] Table 2 Experimental study on the effect of different concentrations of trehalose on goat pox virus culture

[0047] result

[0048] 1. Adding a certain concentration of L-glutamine can significantly accelerate the division of BHK cells and promote their growth. Adding 2mM L-glutamine has the best effect on BHK cells. When divided into bottles at a ratio of 1:8, BHK cells can grow into a dense monolayer in 44 hours, which is 22 hours faster than cells without glutamine. The results are shown in Table 3:

[0049] Table 3 Results of the experiment on the effect of different concentrations of glutamine on BHK cells

[0050] 2. According to the results of healthy cell culture, 2mM L-glutamine was added to the BHK cell growth medium for subculture. Adding a certain concentration of trehalose to the virus maintenance medium can significantly promote the proliferation of goat pox virus, accelerate its pathological process, and increase the content of goat pox virus. Adding 6-8mg / ml trehalose has the best effect. The pathological time is shortened by more than 16 hours compared with no trehalose, and the virus content is increased by more than 1.21 titers (16 times). The trehalose concentration is too high or too low, which is higher than that of the group without trehalose. The results are shown in Table 4:

[0051] Table 4 Experimental results of the effects of different concentrations of trehalose on goatpox virus culture

[0052] Conclusion: Based on the above experiments, adding 2mM L-glutamine during BHK cell culture can significantly increase the cell growth rate, the fastest reaching 22 hours.

[0053] Adding 6-8 mg / ml of trehalose during goatpox virus culture can significantly increase the virus proliferation rate and virus yield, which can be shortened by as fast as 18 hours, and the virus content can be increased by nearly 20 times.

[0054] Therefore, L-glutamine and trehalose can be used for BHK cell culture and goatpox virus culture and proliferation, promoting and improving the yield and quality of goatpox vaccine.

[0055] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A method for increasing the content of goat pox virus, characterized in that: The following steps are involved: Step 1: Healthy cell culture: The frozen BHK cells were revived and subcultured. The basic culture medium was DMEM medium containing 5% newborn calf serum. The cells were divided into 5 groups, with 1 T75 square bottle in each group, and continued to be subcultured. Step 2: Add L-glutamine (0-4 mM) to the culture medium for continued subculturing. Take one bottle and divide it into bottles at a ratio of 1:8 each time. Discard the remaining 7 bottles. When the cells grow into a dense monolayer, digest them with 0.25% trypsin solution and then divide them into bottles for culture. Each group was subcultured for 3 generations, and the time it took for each group of cells to grow into a dense monolayer was observed each time. Step 3: Cultivation of goat pox virus: Add 2 mM L-glutamine to the BHK cell growth medium, digest and disperse the cells according to the conventional method, expand to 40 bottles of cells, divide them into 5 groups, inoculate goat pox virus, each group has 8 bottles, and add 25 ml of virus maintenance solution to each bottle; Step 4: Each group of virus maintenance solution contains 2% newborn calf serum, 2mM L-glutamine, 2% seed virus, and different concentrations of trehalose; Step 5: Discard the growth medium of BHK cells that have grown well, inoculate BHK cells with goatpox virus at 1% to 2% of the total amount of the maintenance medium, add cell maintenance medium, and culture at 37°C. Observe 1 to 2 times a day, harvest when the lesions reach more than 75%, and take samples to test the virus content; Step 6: Dilute the goat pox virus solution in serum-free DMEM medium in 10-fold series, take 3 appropriate dilutions, inoculate 96-well cell plates, inoculate 5 wells of each dilution, 0.1 ml per well, add 0.1 ml of Vero cell suspension containing 200,000 to 300,000 cells per ml to each well, incubate at 37°C, 5% Culture in an incubator for 3 to 5 days, observe the cytopathic effect every day, and make a judgment on the 5th day. Calculate the TCID of goatpox virus based on the number of wells with cytopathic effect 50 .

2. The method for cultivating goatpox virus according to claim 1, characterized in that: The goatpox virus was a weak strain cultured in goatpox testicular cells; Vero cells were used to determine the virus content.

3. The method for cultivating goatpox virus according to claim 2, characterized in that: DMEM medium was used for both production and virus content determination, with 5% newborn calf serum added to the cell culture medium and 2% newborn calf serum added to the maintenance medium.

4. The method for increasing the content of goatpox virus according to claim 1, characterized in that: Adding L-glutamine in step 2 can accelerate the division of BHK cells and promote their growth. Adding 2mM L-glutamine has a good effect on BHK cells. When divided into bottles at a ratio of 1:8, BHK cells can grow into a dense monolayer in 44 hours, which is 22 hours faster than cells without glutamine.

5. The method for increasing the content of goatpox virus according to claim 4, characterized in that: The L-glutamine is prepared separately and stored in a -20°C refrigerator. It is added to the culture solution before use. When the culture solution added with L-glutamine is stored in a 4°C refrigerator for more than 2 weeks, L-glutamine should be added again. The content of L-glutamine in the culture solution is 1-4mM. A 200mM L-glutamine solution is prepared for storage and added to the culture solution when used.

6. The method for increasing the content of goatpox virus according to claim 1, characterized in that: The virus content in step 4 is 10 7.0 TCID 50 / ml.

7. The method for increasing the content of goatpox virus according to claim 6, characterized in that: Adding trehalose to the virus maintenance solution can significantly promote the proliferation of goatpox virus, accelerate its pathological process, and increase the goatpox virus content. Adding 6-8 mg / ml of trehalose has the best effect. The pathological time is shortened by more than 16 hours compared with not adding trehalose, and the virus content is increased by more than 1.21 titers.

8. The method for increasing the content of goatpox virus according to claim 5, characterized in that: The L-glutamine and trehalose are used for BHK cell culture and goat pox virus culture and proliferation, and promote and improve the yield and quality of goat pox vaccine.

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