Method for improving mouse leukemia virus titer and / or stability

Abstract

The invention belongs to the technical field of indication viruses, and particularly relates to a method for improving mouse leukemia virus titer and / or stability. The invention provides an application of mifepristone in improving mouse leukemia virus titer and / or stability. The mifepristone is utilized to integrate the mouse leukemia virus onto the cell genome, so that the amplification of the mouse leukemia virus is completed, the infection efficiency of the mouse leukemia virus is improved, and finally the mouse leukemia virus stock solution with higher titer and better stability is obtained.

Description

Technical Field

[0001] The invention belongs to the technical field of indicator viruses, and in particular relates to a method for improving the titer and / or stability of mouse leukemia virus. Background Art

[0002] Xenotropic murine leukemia virus (X-MuLv) is an important indicator virus and is widely used in the validation of virus clearance / inactivation of biotech therapeutic products produced by CHO. To avoid dilution effect caused by adding virus or changing the properties of the product, the indicator virus titer is required to be as high as possible (>8.0 log 10 ). X-MuLv virus production and purification are particularly important, but its production and efficiency are limited by its inherent instability, which can adversely affect virus production and result in low virus titers. Currently, the industry is working hard to increase the titer of X-MuLv virus, but there are few relevant literature reports on the results.

[0003] Mifepristone is a luteinizing hormone-releasing hormone analog (Gn RH-a), commonly used for female contraception, medical abortion, auxiliary treatment of ectopic pregnancy, and termination of pregnancy. It is used to treat many gynecological research diseases. Currently, there is no record of the effect of mifepristone on the titer and stability of mouse leukemia virus. Summary of the invention

[0004] The purpose of the present invention is to solve the deficiencies of the prior art and improve the titer and stability of mouse leukemia virus.

[0005] In order to achieve the above object, the present invention provides the use of mifepristone in improving the titer and / or stability of mouse leukemia virus.

[0006] Preferably, the mouse leukemia virus comprises a xenotropic mouse leukemia virus.

[0007] The present invention also provides a method for improving the titer and / or stability of mouse leukemia virus, which utilizes mifepristone to amplify the mouse leukemia virus.

[0008] Preferably, the amplification comprises the following steps: The cell culture medium containing mifepristone is mixed and incubated with mouse leukemia virus to obtain a virus inoculum; After the virus inoculum is inoculated into mouse skin fibroblasts, the cells are subcultured using a cell culture medium containing mifepristone, and the culture fluid is collected; the culture fluid contains the mouse leukemia virus.

[0009] Preferably, the virus-inoculated medium containing mifepristone is mixed with mouse leukemia virus for incubation to obtain a virus inoculation solution; After the virus inoculum is inoculated into mouse skin fibroblasts, subculture is performed using a subculture medium containing mifepristone, and the culture fluid is collected; the culture fluid contains the mouse leukemia virus.

[0010] Preferably, the concentration of mifepristone in the inoculation medium containing mifepristone is 0.02-2 μM; the concentration of mifepristone in the passage medium containing mifepristone is 0.02-2 μM.

[0011] Preferably, the number of subcultures is ≥ 3 times.

[0012] Preferably, during the inoculation, the ratio of mouse leukemia virus to mouse skin fibroblasts is 0.01-0.5 MOI: 1.5×10 6 ~3.0×10 6 indivual.

[0013] Preferably, the mixed incubation time is 2-5 hours and the temperature is 36-38°C.

[0014] Preferably, the method of collecting the culture fluid comprises centrifugation; the speed of the centrifugation is 2000-2500 rpm, and the time is 15-20 min.

[0015] Preferably, after collecting the culture fluid, the method further comprises: culturing the culture fluid in a cell culture bottle or a cell factory; and the culturing time is 3 to 9 days.

[0016] Beneficial effects: The present invention provides the use of mifepristone in improving the titer and / or stability of mouse leukemia virus. The present invention utilizes mifepristone to integrate mouse leukemia virus into the cell genome, thereby completing the amplification of mouse leukemia virus, improving the infection efficiency of mouse leukemia virus, and finally obtaining a mouse leukemia virus stock solution with higher titer and better stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0018] Figure 1 This is the cell growth curve of each treatment group in Example 1; Figure 2 The viral titer curves of each treatment group in Example 1; Figure 3 The viral titer curves of each treatment group in Example 2; Figure 4This is the virus titer curve of each treatment group in T75 cell culture flask culture in Example 3; Figure 5 This is the virus titer curve of each treatment group under cell factory culture in Example 3. DETAILED DESCRIPTION

[0019] The invention provides application of mifepristone in improving the titer and / or stability of mouse leukemia virus.

[0020] As an embodiment, the mouse leukemia virus includes a heterotropic mouse leukemia virus. The present invention has no strict requirements on the source of the mifepristone, and conventional purchase is sufficient. As an embodiment, the CAS number of the mifepristone is 84371-65-3. Mifepristone can integrate the mouse leukemia virus into the cell genome, improve the infection efficiency of the mouse leukemia virus, and obtain a mouse leukemia virus stock solution with higher titer and better stability.

[0021] The present invention also provides a method for improving the titer and / or stability of mouse leukemia virus, which utilizes mifepristone to amplify the mouse leukemia virus.

[0022] As an embodiment, the amplification includes the following steps: mixing and incubating a virus-inoculated culture medium containing mifepristone with mouse leukemia virus to obtain a virus inoculation solution; inoculating the virus inoculation solution into mouse skin fibroblasts, performing subculture using a subculture culture medium containing mifepristone, and collecting the culture solution; the culture solution contains the mouse leukemia virus.

[0023] As an embodiment, the present invention mixes and incubates the inoculated culture medium containing mifepristone with mouse leukemia virus to obtain a virus inoculation solution.

[0024] As an embodiment, the concentration of mifepristone in the inoculated medium containing mifepristone is 0.02-2 μM; as another embodiment, the concentration of mifepristone in the inoculated medium containing mifepristone is 0.1-1.5 μM; as another embodiment, the concentration of mifepristone in the inoculated medium containing mifepristone is 0.5-1 μM; as another embodiment, the concentration of mifepristone in the inoculated medium containing mifepristone is 0.5 μM. As an embodiment, the inoculated medium is McCoy's 5A (MC-5A) medium containing 20-28 μM polybrene; as another embodiment, the inoculated medium is McCoy's 5A (MC-5A) medium containing 21.4 μM polybrene. The McCoy's 5A (MC-5A) medium of the present invention does not contain serum and is purchased from Beijing Sino Biological Technology Co., Ltd.

[0025] As an embodiment, the time of the mixed incubation is 2 to 5 hours; as another embodiment, the time of the mixed incubation is 3 to 4 hours. As an embodiment, the temperature of the mixed incubation is 36 to 38°C; as another embodiment, the temperature of the mixed incubation is 37°C. As an embodiment, the mixed incubation uses a CO2 incubator. As an embodiment, the MOI of the virus inoculation solution is 0.01 to 0.5; as another embodiment, the MOI of the virus inoculation solution is 0.1 to 0.25.

[0026] As an embodiment, after obtaining the virus inoculum, the present invention inoculates the virus inoculum into mouse skin fibroblasts, performs subculture using a subculture medium containing mifepristone, and collects the culture fluid; the culture fluid contains the mouse leukemia virus.

[0027] As an embodiment, the concentration of mifepristone in the passage medium containing mifepristone is 0.02-2 μM; as another embodiment, the concentration of mifepristone in the passage medium containing mifepristone is 0.1-1.5 μM; as another embodiment, the concentration of mifepristone in the passage medium containing mifepristone is 0.5-1 μM; as another embodiment, the concentration of mifepristone in the passage medium containing mifepristone is 0.5 μM. As an embodiment, the passage medium can be selected from McCoy's 5a complete medium; the McCoy's 5a complete medium includes a basal medium and 10% FBS, and the basal medium is McCoy's 5A (MC-5A) medium, which does not contain serum and is purchased from Beijing Sino Biological Technology Co., Ltd.

[0028] As an embodiment, the number of subcultures is ≥ 3 times; as another embodiment, the number of subcultures is 3 times. The present invention limits the number of subcultures, and compared with subcultures 0 to 2 times, subcultures ≥ 3 times have the advantage of improving virus infection efficiency and virus replication.

[0029] As an embodiment, during the inoculation, the ratio of mouse leukemia virus to mouse skin fibroblasts is 0.01-0.5 MOI: 1.5×10 6 ~3.0×10 6 As another embodiment, during the inoculation, the ratio of mouse leukemia virus to mouse skin fibroblasts is 0.1~0.25MOI: 2.0×10 6 The present invention limits the inoculation ratio and has the advantage of achieving the best virus infection efficiency.

[0030] In one embodiment, the method for collecting the culture fluid includes centrifugation. In one embodiment, the centrifugal speed is 2000-2500 rpm; in another embodiment, the centrifugal speed is 2200 rpm. In one embodiment, the centrifugal time is 15-20 min; in another embodiment, the centrifugal time is 18 min.

[0031] As an embodiment, after obtaining the culture fluid, it also includes: culturing the culture fluid using a cell culture bottle or a cell factory. As an embodiment, the culturing time is 3 to 9 days; as another embodiment, the culturing time is 5 to 8 days; as another embodiment, the culturing time is 6 to 7 days. As an embodiment, when the culture fluid is cultured using a cell culture bottle, the culturing time is 3 to 8 days; as another embodiment, when the culture fluid is cultured using a cell culture bottle, the culturing time is 5 to 7 days. As an embodiment, the cell culture bottle is a T75 cell culture bottle. As an embodiment, when the culture fluid is cultured using a cell factory, the culturing time is 5 to 8 days; as another embodiment, when the culture fluid is cultured using a cell factory, the culturing time is 7 to 8 days. The present invention has no strict requirements on the culturing method, and the conventional method in the art can be used. The present invention uses a cell culture bottle or a cell factory to culture the culture fluid, which can further improve the virus titer of the mouse leukemia virus and has good stability.

[0032] To further illustrate the present invention, a method for improving the titer and / or stability of mouse leukemia virus provided by the present invention is described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0033] Preparatory Example Prepare 100 mM mifepristone stock solution: weigh 100 mg of mifepristone, add a final volume of 2.33 mL of anhydrous ethanol, and after complete dissolution, filter and sterilize using a 0.2 μm syringe filter and store at 2-8°C for later use.

[0034] Inoculation medium: MC-5A medium containing 21.4 μM polybrene and different concentrations (0 μM, 0.02 μM, 0.1 μM, 0.5 μM, 1 μM, 2 μM) of mifepristone, stored at 2-8°C for later use; Subculture medium: MC-5A medium containing 10% FBS and different concentrations of mifepristone (0μM, 0.5μM, 1μM), stored at 2~8℃ for later use.

[0035] Example 1 (1) Inoculate the cells with xenotropic mouse leukemia virus (X-MuLv, with a virus titer of 5.5 log 10 TCID 50 / mL) and mouse skin fibroblasts (M. dunni) passaged 3 times or more were cultured at 1×10 5 The cells were inoculated into six-well plates at a density of 1000 cells / well and randomly divided into 6 treatment groups for the following treatments: Treatment group 1 (control group): cells were cultured in MC-5A medium (i.e., McCoy's 5A complete medium) containing 10% FBS; Treatment group 2 (0.02 μM mifepristone): cells were cultured in a medium containing 0.02 μM mifepristone; the medium was MC-5A medium supplemented with 10% FBS; Treatment group 3 (0.1 μM mifepristone): cells were cultured in a medium containing 0.1 μM mifepristone; the medium was MC-5A medium supplemented with 10% FBS; Treatment group 4 (0.5 μM mifepristone): cells were cultured in a medium containing 0.5 μM mifepristone; the medium was MC-5A medium supplemented with 10% FBS; Treatment group 5 (1 μM mifepristone): cells were cultured in a medium containing 1 μM mifepristone; the medium was MC-5A medium supplemented with 10% FBS; Treatment group 6 (2 μM mifepristone): cells were cultured in a medium containing 2 μM mifepristone; the medium was MC-5A medium supplemented with 10% FBS; (2) Step (1) Each treatment group was cultured at 37°C and 5% CO2. After inoculation, the viral supernatant was harvested every day, and the cells were digested for cell counting and TCID was calculated. 50 The value was used to determine the virus titer and the effect of mifepristone on the growth of M. dunni cells and virus amplification. The results are shown in Tables 1-2 and Figure 1~2 shown.

[0036] Table 1 Cell density under different concentrations of mifepristone (10 5 pcs / mL)

[0037] Table 2 Virus titers (log 10 TCID 50 / mL)

[0038] According to Table 1~2 and Figure 1~2It can be seen that different concentrations of mifepristone have little effect on the growth of infected M. dunni cells, and the addition of 0.5 μM and 1 μM mifepristone showed a virus amplification effect that was no less than that of the control group.

[0039] Example 2 The addition stage and optimal concentration of mifepristone (1) M. dunni cells were inoculated into T75 cell culture flasks. On the second day, when the cell confluence was about 80%, they were randomly divided into five treatment groups and treated as follows: Treatment group 1 (control group): Xenotropic murine leukemia virus (X-MuLv) was diluted to an MOI of 0.25 using the inoculation medium without mifepristone in the preparation to obtain a virus dilution solution; the virus dilution solution was inoculated into a T75 cell culture flask containing M.dunni cells and incubated in a CO2 incubator; after 3 hours, the inoculation medium was added to the T75 cell culture flask; the cells were passaged 3 times using the passage medium without mifepristone in the preparation; Treatment group 2 (Inf-0.5 μM mifepristone): Xenotropic murine leukemia virus (X-MuLv) was diluted to an MOI of 0.25 using the inoculation medium containing 0.5 μM mifepristone in the preparation to obtain a virus dilution; the virus dilution was inoculated into a T75 cell culture flask containing M.dunni cells and incubated in a CO2 incubator; after 3 hours, the T75 cell culture flask was supplemented with an inoculation medium containing 0.5 μM mifepristone; the cells were passaged 3 times using the passage medium without mifepristone in the preparation; Treatment group 3 (I+P-0.5μM mifepristone): Xenotropic murine leukemia virus (X-MuLv) was diluted to an MOI of 0.25 using the inoculation medium containing 0.5μM mifepristone in the preparation to obtain a virus dilution; the virus dilution was inoculated into a T75 cell culture flask containing M.dunni cells and incubated in a CO2 incubator; after 3 hours, the T75 cell culture flask was supplemented with an inoculation medium containing 0.5μM mifepristone; the cells were passaged 3 times using the passage medium containing 0.5μM mifepristone in the preparation; Treatment group 4 (Inf-1μM mifepristone): Xenotropic murine leukemia virus (X-MuLv) was diluted to an MOI of 0.25 using the inoculation medium containing 1μM mifepristone in the preparation to obtain a virus dilution; the virus dilution was inoculated into a T75 cell culture flask containing M.dunni cells and incubated in a CO2 incubator; after 3 hours, the T75 cell culture flask was supplemented with an inoculation medium containing 1μM mifepristone; the cells were passaged 3 times using the passage medium without mifepristone in the preparation; Treatment group 5 (I+P-1μM mifepristone): Xenotropic murine leukemia virus (X-MuLv) was diluted to an MOI of 0.25 using the inoculation medium containing 1μM mifepristone in the preparation to obtain a virus dilution; the virus dilution was inoculated into a T75 cell culture flask containing M.dunni cells and incubated in a CO2 incubator; after 3 hours, the T75 cell culture flask was supplemented with an inoculation medium containing 1μM mifepristone; the cells were passaged three times using the passage medium containing 1μM mifepristone in the preparation.

[0040] (2) Step (1) After three passages, the virus culture supernatant of each treatment group was harvested in six batches. The virus solution was centrifuged at 2000-2500 rpm for 15-20 min, the supernatant was collected, and the TCID 50 The virus titer was detected by the method, and the results are shown in Table 3 and Figure 3 shown.

[0041] Table 3 Virus titers in different treatment groups (log 10 TCID 50 / mL)

[0042] Among them, “ / ” means no significant difference analysis was performed, ns means no significant difference, * P <0.05.

[0043] According to Table 3 and Figure 3 It can be seen that the third treatment group (addition of 0.5 μM mifepristone during inoculation and the third passage) can significantly increase the X-MuLv virus titer.

[0044] Example 3 (1) M. dunni cells were inoculated into T75 cell culture flasks. On the second day, when the cell confluence was about 80%, they were randomly divided into two treatment groups and treated as follows: Treatment group 1 (control group): Xenotropic murine leukemia virus (X-MuLv) was diluted to an MOI of 0.25 using the inoculation medium without mifepristone in the preparation to obtain a virus dilution solution; the virus dilution solution was inoculated into a T75 cell culture flask containing M.dunni cells and incubated in a CO2 incubator; after 3 hours, the inoculation medium was added to the T75 cell culture flask; the cells were passaged 3 times using the passage medium without mifepristone in the preparation; Treatment group 2 (0.5 μM mifepristone): Xenotropic murine leukemia virus (X-MuLv) was diluted to an MOI of 0.25 using the inoculation medium containing 0.5 μM mifepristone in the preparation to obtain a virus dilution; the virus dilution was inoculated into a T75 cell culture flask containing M. dunni cells and incubated in a CO2 incubator; after 3 hours, the T75 cell culture flask was supplemented with an inoculation medium containing 0.5 μM mifepristone; and the cells were passaged three times using the passage medium containing 0.5 μM mifepristone in the preparation.

[0045] (2) Step (1) After three passages, each treatment group was cultured in a T75 cell culture flask and a cell factory to amplify the X-MuLv virus. During the amplification process, the virus supernatant was harvested and the TCID was calculated. 50 The virus titer was determined by the value, and the results are shown in Tables 4~5 and Figures 4 and 5 shown.

[0046] Table 4 Virus titers in different treatment groups - T75 (log 10 TCID 50 / mL)

[0047] Among them, “ / ” means no significant difference analysis was performed, * P <0.05.

[0048] Table 5 Virus titer in different treatment groups - cell factory (log 10 TCID 50 / mL)

[0049] Among them, “ / ” indicates that no significant difference analysis was performed, ** P <0.01.

[0050] According to Tables 4~5 and Figures 4 and 5 It can be seen that mifepristone significantly promoted the increase in X-MuLv virus titer regardless of whether T75 cell culture flask or cell factory culture was used.

[0051] According to the above content, it can be seen that mifepristone can improve the infection efficiency of mouse leukemia virus and improve the virus titer and stability of mouse leukemia virus.

[0052] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of mifepristone in improving the titer and / or stability of mouse leukemia virus.

2. The use according to claim 1, characterized in that: The mouse leukemia virus includes a xenotropic mouse leukemia virus.

3. A method for increasing the titer and / or stability of mouse leukemia virus, characterized in that: Amplification of mouse leukemia virus using mifepristone.

4. The method according to claim 3, characterized in that The amplification comprises the following steps: The virus inoculation medium containing mifepristone is mixed and incubated with mouse leukemia virus to obtain a virus inoculation solution; After the virus inoculum is inoculated into mouse skin fibroblasts, subculture is performed using a subculture medium containing mifepristone, and the culture fluid is collected; the culture fluid contains the mouse leukemia virus.

5. The method according to claim 4, characterized in that The concentration of mifepristone in the inoculation medium containing mifepristone is 0.02-2 μM; the concentration of mifepristone in the passage medium containing mifepristone is 0.02-2 μM.

6. The method according to claim 4, characterized in that The number of subcultures is ≥ 3 times.

7. The method according to claim 4, characterized in that During the inoculation, the ratio of mouse leukemia virus to mouse skin fibroblasts was 0.01-0.5 MOI: 1.5×10 6 ~3.0×10 6 indivual.

8. The method according to claim 4, characterized in that The mixed incubation time is 2-5 hours, and the temperature is 36-38°C.

9. The method according to claim 4, characterized in that The method of collecting the culture fluid includes centrifugation; the speed of the centrifugation is 2000-2500 rpm, and the time is 15-20 min.

10. The method according to any one of claims 4 to 9, characterized in that: After collecting the culture fluid, the method further comprises: culturing the culture fluid in a cell culture bottle or a cell factory; the culturing time is 3 to 9 days.

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