Method for improving expression quantity of bovine rotavirus

Abstract

The invention relates to a method for improving the expression quantity of viruses, in particular to a method for improving the expression quantity of bovine rotaviruses. The method comprises the following steps: adding Marc-145 cells into a cell culture bottle or a cell culture plate acted by polylysine for subculture, and then inoculating bovine rotavirus for culture. According to the method, the Marc-145 cells are cultured by using the cell culture bottle or the cell culture plate acted by the polylysine, so that the tolerance degree of the Marc-145 cells to high-concentration trypsin is improved, and a proper pancreatin tolerance concentration value is screened out. Furthermore, the bovine rotavirus is inoculated into a DMEM culture medium containing Marc-145 cells and high-concentration trypsin, so that the expression quantity of the bovine rotavirus is improved, and the virus titer of virus expression is remarkably improved.

Description

Technical Field

[0001] The present invention relates to a method for increasing the expression amount of a virus, and in particular to a method for increasing the expression amount of a bovine rotavirus. Background Art

[0002] Bovine rotavirus (BRV) is a virus belonging to the genus Rotavirus of the family Reoviridae. It is one of the main pathogens causing diarrhea in calves, which can cause serious economic losses to the cattle industry. Bovine rotavirus is transmitted through the digestive tract, with an incubation period of about 18 to 96 hours. It mainly infects calves within 3 months of age, with more cases in spring and autumn. The virus exists in the intestines and mainly attacks the small intestine, especially the jejunum and ileum, causing thinning of the intestinal wall, liquefaction of the contents, and atrophy of the small intestinal villi. Calves infected with bovine rotavirus mainly show diarrhea and dehydration, with white or grayish white or yellow-brown feces, which may be sticky or watery, and sometimes contain intestinal mucosa and undigested curds. Symptoms such as depression, loss of appetite, and normal or slightly high body temperature may also occur.

[0003] Patent CN110055225A discloses a method for increasing the yield of rotavirus, which is to infect tissue culture cells with rotavirus, culture the infected cells in a culture system, and thus harvest the rotavirus. In which, 2.0-10.0 μg / ml pancreatin is added to the culture system continuously or multiple times. Chinese patent CN 1306961C provides a method for preparing an inactivated rotavirus vaccine, which adds 1-1.5 μg / ml pancreatin to a cell culture system for culturing tissue cells infected with rotavirus.

[0004] The prior art has not found a method of increasing the tolerance concentration of cells to trypsin by using reagents, and then performing virus inoculation culture in a high-concentration trypsin culture system to improve the expression of bovine rotavirus and increase the virus titer. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a method for increasing the expression amount of bovine rotavirus in view of the deficiencies of the prior art.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0007] A method for increasing the expression of bovine rotavirus comprises adding Marc-145 cells to a cell culture bottle or a cell culture plate treated with poly-lysine, gelatin, collagen or fibronectin for subculture, and then inoculating bovine rotavirus for culture.

[0008] Preferably, Marc-145 cells are added to a cell culture flask or a cell culture plate treated with 0.2-1.5% poly-lysine, 0.1% gelatin, 0.1% collagen or 10 μg / mL fibronectin for subculture, and then inoculated with bovine rotavirus for culture.

[0009] Preferably, Marc-145 cells are added to a cell culture flask or a cell culture plate treated with 0.2-1.5% poly-lysine for subculture, and then inoculated with bovine rotavirus for culture.

[0010] A more preferred poly-lysine concentration is 1 to 1.5%, and a most preferred poly-lysine concentration is 1%.

[0011] Wherein, the conditions for the action of poly-lysine, gelatin, collagen or fibronectin are: incubation at 4°C for more than 2 hours or incubation at 37°C for more than 30 minutes.

[0012] In some embodiments of the present invention, the tolerance of Marc-145 cells to trypsin is improved after being treated with poly-lysine, gelatin, collagen or fibronectin.

[0013] Specifically, when the gelatin concentration is 0.1% (w / v), the tolerance concentration of Marc-145 cells to trypsin is 5-10 μg / ml; when the collagen concentration is 0.1% (w / v), the tolerance concentration of Marc-145 cells to trypsin is 5-10 μg / ml; when the fibronectin concentration is 10 μg / ml, the tolerance concentration of Marc-145 cells to trypsin is 5-10 μg / ml; when the poly-lysine concentration is 0.2-1.5% (w / v), the tolerance concentration of Marc-145 cells to trypsin is 5-15 μg / ml.

[0014] The Marc-145 cells are obtained by digesting the Marc-145 cells grown into a dense monolayer with 2.5 g / L trypsin at room temperature.

[0015] The addition density of Marc-145 cells is 2-3×10 5 Pieces / ml.

[0016] The inoculation of bovine rotavirus specifically involves inoculating the bovine rotavirus into a culture medium containing Marc-145 cells grown into a dense monolayer obtained by subculture and trypsin.

[0017] Specifically, the inoculation amount is 10-25% of the volume of the culture medium.

[0018] Specifically, the culture medium is DMEM culture medium.

[0019] The culture conditions are as follows: incubate at 36-38°C, 5% CO2 for 1-2 hours, discard the liquid, add DMEM medium containing 5-15 μg / ml trypsin, and culture at 36-38°C, 5% CO2 for 48-72 hours.

[0020] In some embodiments of the present invention, the virus titer of bovine rotavirus was increased to 10 by inoculating bovine rotavirus into DMEM medium containing Marc-145 cells grown into a dense monolayer obtained by subculture and high concentration of trypsin. 8.0 TCID 50 / ml or above.

[0021] Beneficial effects: The present invention utilizes poly-lysine, gelatin, collagen or fibronectin to culture Marc-145 cells in cell culture flasks or cell culture plates, thereby improving the tolerance of Marc-145 cells to high concentrations of trypsin and screening out a suitable tolerance concentration of trypsin. Furthermore, by inoculating bovine rotavirus into a DMEM culture medium containing Marc-145 cells and high concentrations of trypsin, the effect of improving the virus yield is achieved, the expression of bovine rotavirus is increased, and the virus titer of the virus expression is significantly increased to 10 8.0 TCID 50 / ml, and after P5, the virus content was nearly 1 titer higher than that of the virus in the untreated cell flask culture cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0023] Figure 1 Figure 2 shows CPE lesions in normal Marc-145 cells and cells infected with bovine rotavirus.

[0024] Figure 2 The immunofluorescence results of immunofluorescence detection are shown in Figure 1. Among them, a represents the neutralization test group; b represents the virus control group; and c represents the healthy cell control group. DETAILED DESCRIPTION

[0025] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0026] In the following examples, the Marc-145 cells were derived from the China Veterinary Drug Administration; the gelatin and collagen were purchased from Sigma; the poly-lysine was purchased from Shanghai Jinyuan Biotechnology; the fibronectin was purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.; the trypsin was purchased from Gibco; and the FITC secondary antibody was purchased from abcam.

[0027] In the following examples, the anti-bovine rotavirus specific serum was prepared by Tiankang Biopharmaceutical Co., Ltd. The preparation process is as follows: 2 healthy susceptible calves aged 7 days were injected with 2 ml of inactivated bovine rotavirus vaccine (the antigen content before inactivation was not less than 10 7.5 TCID 50 / ml), and were isolated and raised under the same conditions. The second immunization was carried out 21 days after the immunization. Blood was collected 14 days after the second immunization, and serum was separated for immunofluorescence detection to obtain anti-bovine rotavirus specific serum.

[0028] In the following examples, w / v refers to g / mL unless otherwise specified.

[0029] Example 1: Reagent screening test

[0030] (1) Reagent screening:

[0031] Add 0.5-1.0 ml of 0.1% gelatin (w / v), 1% poly-lysine (w / v), 0.1% collagen (w / v), and 10 μg / ml fibronectin (needed to cover the bottom of the well) to the culture wells of the cell culture plate, and incubate at 4°C for more than 2 h or at 37°C for more than 30 min. Take Marc-145 cells that have grown into a dense monolayer for 3 days, digest the cells with 2.5 g / L trypsin at room temperature, and then add 2-3×10 5 The cells were subcultured in 6-well plates at a cell density of 100 μg / ml. The Marc-145 cells that had grown into a dense monolayer after 3 days of culture were added with a gradient of trypsin at a final concentration of 5, 10, 15, and 20 μg / ml. The cells were cultured in a carbon dioxide incubator at 37°C and 5% CO2, and the cell status was observed every day. The cells without the addition of reagents were used as control cells.

[0032] Table 1 Reagent screening results

[0033]

[0034] The results are shown in Table 1. When Marc-145 cells were cultured on a 1% poly-lysine cell culture plate, the cells could tolerate a maximum trypsin concentration of 15 μg / ml. When the trypsin concentration was not higher than 15 μg / ml, the cells grew normally without shrinking or falling off. The cells coated with 0.1% gelatin, 0.1% collagen, 10 μg / ml fibronectin, and control cells could tolerate a trypsin concentration of 10 μg / ml. When the trypsin concentration was lower than 10 μg / ml, the cells grew normally without shrinking or falling off.

[0035] (2) Confirmation of poly-lysine concentration

[0036] Add 0.5-1.0 ml of 0.2%, 0.5%, 1%, 1.5% (w / v) poly-lysine to the culture wells of the cell culture plate (need to cover the bottom of the well), incubate at 4°C for more than 2 hours or at 37°C for more than 30 minutes. Take Marc-145 cells that have grown into a dense monolayer for 3 days, digest the cells with 2.5 g / L trypsin at room temperature, and then add 2-3 × 10 5 The cells were subcultured in 6-well plates at a cell density of 100 μg / ml. Marc-145 cells that had grown into a dense monolayer after 3 days of culture were added with a gradient of trypsin at a final concentration of 5, 10, 15, and 20 μg / ml. The cells were cultured in a carbon dioxide incubator at 37°C and 5% CO2, and the cell status was observed every day.

[0037] Table 2: Poly-L-lysine concentration screening results

[0038]

[0039]

[0040] The results are shown in Table 2. When Marc-145 cells were cultured on 1% and 1.5% poly-lysine cell culture plates, the cells could tolerate a maximum trypsin concentration of 15 μg / ml. When the trypsin concentration was not higher than 15 μg / ml, the cells grew normally without shrinking or falling off. When the cells cultured on 0.2% and 0.5% poly-lysine cell culture plates and control cells could tolerate a trypsin concentration of 10 μg / ml. When the trypsin concentration was lower than 10 μg / ml, the cells grew normally without shrinking or falling off.

[0041] Example 2: A method for increasing the expression of bovine rotavirus

[0042] 1. Cell culture

[0043] Add 1% poly-lysine to the culture wells of the cell flask or cell culture plate (need to cover the bottom of the flask / well), incubate at 4°C for more than 2 hours or at 37°C for more than 30 minutes. Take Marc-145 cells that have grown into a dense monolayer for 3 days, digest the cells with 2.5g / L trypsin at room temperature, and then add 2-3×10 5 The cell density was 100 cells / ml for subculture in 6-well plates or cell flasks.

[0044] 2. Treatment of diseased materials

[0045] Aseptically collect the small intestinal tissue or feces of the diarrheal calf, cut the small intestinal tissue into pieces in a biosafety cabinet, add DMEM culture medium, and grind it into a paste. Transfer it to a sterile centrifuge tube; centrifuge it at 4℃10000rpm for 20min, take the supernatant, sterilize and filter it with a 0.22μm filter membrane to obtain the tissue bovine rotavirus liquid for later use.

[0046] 3. Virus culture

[0047] Add 5 ml of maintenance solution containing trypsin (DMEM + 15 μg / ml trypsin) to Marc-145 cells growing in dense monolayer (cells cultured in cell flasks with 1% poly-lysine); add 5 ml of maintenance solution containing trypsin (DMEM + 10 μg / ml trypsin) to Marc-145 cells growing in dense monolayer (cells cultured in cell flasks without action). Inoculate the prepared bovine rotavirus liquid in a volume ratio of 10-25% respectively, place incubator at 36-38°C, 5% CO2 for 1-2 hours, discard the liquid, add 5 ml of DMEM culture medium containing 15 μg / ml trypsin, and culture in a 36-38°C, 5% CO2 incubator for 48-72 hours for harvest. Use unacted Marc-145 cells for virus culture as a control.

[0048] The results are shown in Table 3 and Figure 1 As shown, Marc-145 cells (cells cultured in cell flasks treated with 1% poly-lysine) growing in dense monolayers were used for virus subculture. After 48 to 72 hours of culture, cytopathic effects appeared at the P2 generation of blind virus passage, with cells showing rounding, exposed cytoplasm, and shedding. Moreover, the virus content after the P5 generation was nearly 1 titer higher than that of the virus cultured in cell flasks without the treatment of cells.

[0049] Table 3 Bovine rotavirus passage virus content determination results (unit: log10 TCID 50 / ml)

[0050]

[0051] Example 3: Identification of viruses

[0052] (1) Specificity: Dilute 0.03 ml of virus with 10 ml of serum-free DMEM medium to 200 TCID 50 / 0.1ml, mixed with an equal amount of anti-bovine rotavirus specific serum to obtain a neutralizing solution, which was placed at 37°C for 1 hour as a neutralization test group. The neutralizing solution was then inoculated into a cell culture plate that had grown into a complete monolayer of Marc-145 cells, and the cell pathological changes were observed for 5 to 7 days after inoculation. At the same time, a virus control group and a healthy cell control group were set up.

[0053] The results showed that the neutralization test group had no cytopathic effect, the virus control group had all cytopathic effect characterized by cell shrinkage, and the cell control group was normal with no cytopathic effect, indicating that bovine rotavirus can be completely neutralized by anti-bovine rotavirus specific serum.

[0054] (2) Immunofluorescence detection: 0.03 ml of virus was diluted to 200 TCID with 10 ml of serum-free DMEM medium. 50 / 0.1ml, mixed with an equal amount of anti-bovine rotavirus specific serum to obtain a neutralizing solution, placed the neutralizing solution at 37°C for 1 hour, and then inoculated the neutralizing solution into 10 wells of a 96-well cell culture plate that had grown into a complete monolayer of Marc-145 cells, 0.1ml / well; at the same time, set up a virus control group and a healthy cell control group, 10 wells each, the virus control group was inoculated with 0.1ml / well of the virus, and the healthy cell control group was inoculated with 0.1ml / well of serum-free DMEM. The 96-well cell culture plate was placed in a carbon dioxide incubator at 36-38°C and 5% CO2 for 5-7 days. Washed 3 times with PBST buffer (pH7.2). After fixing with 80% pre-cooled acetone for 10 minutes, washed 3 times with PBST buffer (pH7.2). Add anti-bovine rotavirus specific serum (1:500), incubate at 37°C for 1 hour, and wash 3 times with PBST buffer (pH7.2). Add FITC secondary antibody (1:200), incubate at 37℃ for 1 hour, and wash 3 times with PBST buffer (pH7.2). Finally, add 50μl PBST buffer (pH7.2) to each well. Observe with an inverted fluorescence microscope. After adding fluorescent secondary antibody, the results are as follows Figure 2 As shown, no specific fluorescence appeared in the neutralization test group, specific fluorescence appeared in the virus control group, and no fluorescence appeared in the healthy cell control group.

[0055] (3) Toxicity to calves: 3.18 ml of virus was diluted with saline to a virus content of 10 5.0 TCID 50 / ml of virus liquid was inoculated into 5 healthy susceptible calves of 7 days old, with 1ml / head given orally. After 7 consecutive days of observation, 4 calves infected with bovine rotavirus became ill, with symptoms of diarrhea and sticky milky white or yellowish white feces. The feces of more seriously ill cows contained mucus or blood.

[0056] (4) Immunogenicity: 10 healthy susceptible calves aged 7 days were used, and 5 of them were injected intramuscularly with 2 ml of inactivated bovine rotavirus vaccine (the antigen content before inactivation was not less than 10 7.5 TCID 50 / ml), and the other 5 cows were not injected as controls and were isolated and raised under the same conditions. 21 days after immunization, the cows and the control cows were orally given 1ml of virus (virus content 10 5.0 TCID 50 / ml), and observed for 7 consecutive days. The results showed that 4 heads of control cattle in each challenge group were sick, while 4 heads of immunized cattle in each immunized group were protected.

[0057] (5) Purity: According to the current Appendix of the Chinese Veterinary Pharmacopoeia (Volume 3), the products were tested and found to be free of bacteria, mold, mycoplasma and exogenous virus contamination. The results showed that the viruses were all pure.

[0058] The present invention provides a method and method for increasing the expression of rotavirus. There are many methods and approaches to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the protection scope of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. A method for increasing the expression level of bovine rotavirus, characterized in that: Marc-145 cells are added to cell culture flasks or cell culture plates treated with poly-lysine, gelatin, collagen or fibronectin for subculture, and then inoculated with bovine rotavirus for culture.

2. The method according to claim 1, characterized in that Marc-145 cells are added to a cell culture bottle or a cell culture plate treated with 0.2-1.5% poly-lysine, 0.1% gelatin, 0.1% collagen or 10 μg / ml fibronectin for subculture, and then inoculated with bovine rotavirus for culture.

3. The method according to claim 2, characterized in that Marc-145 cells are added to a cell culture bottle or a cell culture plate treated with 0.2-1.5% poly-lysine for subculture, and then inoculated with bovine rotavirus for culture; preferably, the concentration of poly-lysine is 1-1.5%.

4. The method according to claim 1, characterized in that: The conditions for the action of poly-lysine, gelatin, collagen or fibronectin are: incubation at 4°C for more than 2 hours or incubation at 37°C for more than 30 minutes.

5. The method according to claim 1, characterized in that The Marc-145 cells are obtained by digesting the Marc-145 cells grown into a dense monolayer with 2.5 g / L trypsin at room temperature.

6. The method according to claim 1, characterized in that The addition density of the Marc-145 cells is: 2-3×10 5 Pieces / ml.

7. The method according to claim 1, characterized in that The inoculation of bovine rotavirus specifically involves inoculating the bovine rotavirus into a culture medium containing Marc-145 cells grown into a dense monolayer obtained by subculture and trypsin.

8. The method according to claim 7, characterized in that The inoculation amount is 10-25% of the volume of the culture medium; the culture medium is DMEM culture medium.

9. The method according to claim 1, characterized in that: The culture conditions are as follows: incubate at 36-38°C and 5% CO2 for 1-2 hours, discard the liquid, add DMEM culture medium containing 5-15 μg / ml trypsin, and culture at 36-38°C and 5% CO2 for 48-72 hours.

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