Method for culturing Japanese encephalitis virus P3 strain on Vero cell and characteristic research

Abstract

The invention discloses a culture method and characteristic research of a Japanese encephalitis virus P3 strain on a Vero cell, and belongs to the field of bioengineering. The invention provides the preparation method of the Vero cell carrying the Japanese encephalitis virus P3 strain, the virus titer of the obtained cell reaches 7.44 pfu / mL, and the cell has high hereditary stability.

Description

Technical Field

[0001] The invention relates to a culture method of Japanese encephalitis virus P3 strain on Vero cells and characteristic research, and belongs to the field of bioengineering. Background Art

[0002] Japanese encephalitis virus is abbreviated as JE virus. It is the pathogen that causes Japanese encephalitis. The virus particles are spherical, the nucleic acid is single-stranded positive-strand RNA, the outer layer has an envelope, and the surface of the envelope has hemagglutinin. It can proliferate in animals, chicken embryos and sensitive tissue culture cells. Pigs are the intermediate hosts of JE virus, and mosquitoes are important vectors of JE virus. When a person is bitten by a mosquito carrying the virus, the JE virus enters the human body, proliferates in phagocytic cells such as vascular endothelial cells, lymph nodes, liver, spleen, etc., and reaches the brain through the blood circulation to cause inflammation.

[0003] In recent years, some northern regions that previously had a low prevalence of JE have seen an epidemic of JE mainly among adults. The reason for this is thought to be that some adults in the northern low-prevalence regions who had not been vaccinated or naturally infected are in a susceptible state. If changes in local climate, precipitation, and livestock breeding environment factors such as pigs and cattle create conditions conducive to the spread of the JE virus, these susceptible adults may be at risk of contracting JE if they are bitten by Culex mosquitoes carrying the virus (mainly Culex tritaeniorhynchus).

[0004] There is no specific drug to treat Japanese encephalitis, and the effect of controlling its spread from the environment is not ideal. Immunization with Japanese encephalitis vaccine is the most economical and effective measure to control Japanese encephalitis. Preventing Japanese encephalitis is extremely important for protecting children's health. After Japanese encephalitis vaccine is administered in my country, the protection rate of the population can reach more than 90%.

[0005] BHK-21 cell line, C6 / 36 cell line and chicken embryo fibroblasts are commonly used sensitive cells. Virus proliferation in cells causes cell rounding, increased particles, cell shedding and other cytopathic effects. Infectious viruses are contained in the culture supernatant, and specific antigens can be detected on the cell membrane in the cytoplasm. Vero cells can also be used as a culture carrier of Japanese encephalitis virus, but the prior art still needs to improve the adaptability of Japanese encephalitis virus P3 strain on Vero cells. Summary of the invention

[0006] The purpose of the invention is to improve the adaptability of Japanese encephalitis virus P3 strain on Vero cells and cultivate a high-titer adaptable strain on the cell line.

[0007] The present invention provides a method for adaptive culture and detection of Japanese encephalitis virus P3 strain on Vero cells, which comprises the following steps in sequence:

[0008] S1: Vero cells cultured in commercial MEM medium in cell culture flasks;

[0009] S2: Inoculation of Japanese encephalitis virus P3 strain on Vero cells, MOI: 0.01;

[0010] S3: Virus culture conditions: Commercial MEM culture medium supplemented with 2% fetal bovine serum; culture conditions: 36.5℃~37.5℃, 5% CO 2 ; Cultivation time: 48 to 72 hours.

[0011] S4: The virus titer of the Japanese encephalitis virus P3 strain is detected after each passage, and the method includes: plaque assay.

[0012] The present invention also provides a cell strain expressing Japanese encephalitis virus prepared by the method.

[0013] The present invention also provides application of the method or the cell strain in preparing Japanese encephalitis vaccine.

[0014] The present invention inoculates the Japanese encephalitis virus P3 strain on Vero cells for adaptive passage, and the experimental results show that after the Vero cells are cultured in step S1, the Japanese encephalitis virus P3 strain can grow and reproduce well on the Vero cells under the culture conditions of step S3, and the virus titer changes with the culture time and presents a parabolic trend, and after 60 hours of culture, the virus titer reaches 7.44pfu / mL. The whole genome sequencing of the domesticated strain is performed, and the strain has high genetic stability and the main antigen epitopes are unchanged.

[0015] Beneficial effects:

[0016] The invention provides a method for preparing Vero cells carrying Japanese encephalitis virus P3 strain. The obtained cell virus titer reaches 7.44 pfu / mL and has high genetic stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The pathological characteristics of Vero cells inoculated with Japanese encephalitis virus (P3) at different time periods;

[0018] Figure 2 This is the growth curve of Japanese encephalitis virus (P3) with different titers in Vero cells at different inoculation times. DETAILED DESCRIPTION

[0019] The Vero cells (CBP60971) involved in the following examples were stored in our laboratory for a long time and were originally purchased from ATCC; the Japanese encephalitis virus P3 strain was isolated and preserved by our staff, and its nucleotide sequence is shown in Genebank U47032.1.

[0020] The Vero cell culture medium involved in the following examples is:

[0021] Complete medium: Commercial MEM medium supplemented with 1% PS (penicillin-streptomycin) and 10% FBS (fetal bovine serum);

[0022] Maintenance medium: Commercial MEM medium was supplemented with PS and FBS, with the concentrations of 1% PS and 2% FBS, respectively.

[0023] Example 1: Japanese encephalitis virus P3 strain passage adaptation method

[0024] Vero cells were inoculated in culture flasks containing complete medium and incubated in an atmosphere of 5% CO. 2 , 37℃ incubator. When the cells grow to the entire culture bottle, they can be passaged. Discard the culture medium, add 4-6mL PBS, shake the cell bottle horizontally to rinse the remaining culture medium in the bottle, discard PBS, add 1-2mL EDTA-trypsin, and observe the degree of cell digestion under a microscope. After the cell gap increases and the cells become round and shrink, gently pour out the trypsin, add an appropriate amount of complete culture medium to terminate digestion, and gently blow the cell suspension to make the cells distributed individually, divide the cell suspension into new cell culture bottles, and place the cells in 5% CO 2 , cultured in a 37°C incubator.

[0025] Cytopathic effect observation:

[0026] When the cells grew to 90% confluence in the cell flask, the Japanese encephalitis virus P3 was inoculated into the Vero cells at an MOI of 0.01. 2 After incubation at 37°C for 1 hour, the maintenance medium was added. The cell lesions were observed and recorded every 12 hours. Figure 1 After the cells were inoculated with the virus at MOI 0.01, the cytopathic effect was not obvious after 24 hours, but began to appear after 36 hours, and became obvious after 48 hours, with some cells showing rounded lesions. After 72 hours of virus inoculation, more than 50% of the cells showed rounded lesions and some lesioned cells fell off.

[0027] Virus growth curve assay:

[0028] The P3 strain with determined virus titer was used to inoculate Vero cells with a virus amount of MOI = 0.01-0.05. After the virus adsorbed to the cells for 1 hour, the cells were washed with PBS and supplemented with cell maintenance medium. The cells and supernatant were collected at 0, 12, 24, 36, 48, 60, 72, 84 and 96 hours after infection, respectively. After repeated freezing and thawing for 3 times, the supernatant was collected by centrifugation at 4°C and 2500×g for 15 minutes. The supernatant was titrated by plaque assay, and the growth curve of the virus on different cell lines was plotted using GraphPad Prism 8.0 software (GraphPad Software) ( Figure 2 ), with virus titer as the ordinate and hours after inoculation (h) as the abscissa. Each time point was tested three times independently to calculate the average value, as shown in Table 1. Two-way ANOVA was used to perform statistical tests on the titers between different groups. The results of the virus growth curve showed that the highest virus titer was collected 60 hours after the cells were inoculated with MOI = 0.01 virus titer, and the highest virus titer was 1×10 7.44 pfu / mL, while the virus titer collected 48 hours after the cells were inoculated with MOI = 0.05 virus was the highest, with a virus titer of 1×10 7.14 pfu / mL. Experiments show that the best titer for cell inoculation is the virus with an MOI of 0.01.

[0029] Table 1 Virus titers of different titers of Japanese encephalitis virus (P3) in Vero cells at different inoculation times

[0030] Hours post infection 0.01MOI titer (pfu / mL) 0.05MOI titer (pfu / mL) 0 3.52 3.68 12 3.15 3.92 24 6.53 6.65 36 7.30 7.01 48 7.40 7.14 60 7.44 6.88 72 6.95 6.27 84 5.23 4.62 96 4.08 3.83

[0031] Take a cell culture dish inoculated with the virus at a titer of MOI = 0.01 for 60 hours, use a pipette to remove the supernatant, use a viral RNA extraction kit to extract the viral nucleic acid, use a reverse transcription kit to reverse transcribe the nucleic acid into cDNA, and then perform full gene sequence determination. The result is shown in SEQ ID No.1. Compared with the original Japanese encephalitis virus P3 strain, it has higher genetic stability and the main antigenic epitopes have not changed.

[0032] In summary, the method of the present invention can successfully improve the adaptability of the Japanese encephalitis virus P3 strain on Vero cells and cultivate a high-titer adapted strain on Vero cells.

[0033] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for adapting the P3 strain of Japanese encephalitis virus to culture on Vero cells, characterized in that: The following steps are involved: (1) Cultivating Vero cells; (2) inoculating the Japanese encephalitis virus P3 strain into the Vero cells cultured in step (1) at an MOI of 0.01-0.05 and incubating for at least 1 hour; (3) After the incubation in step 2, the cells are supplemented with culture medium and the culture is continued.

2. The method according to claim 1, characterized in that Step (2) inoculating the Japanese encephalitis virus P3 strain into the cultured Vero cells at an MOI of 0.

01.

3. The method according to claim 1, characterized in that: Step (1) Vero cells are cultured in MEM medium containing serum.

4. The method according to claim 1, characterized in that: Step (2) is incubated under the conditions of 5% CO2 and 37°C.

5. The method according to claim 1, characterized in that The culture medium in step (3) includes but is not limited to MEM culture medium containing serum.

6. The method according to claim 5, characterized in that The serum content in the culture medium is at least 2%.

7. The method according to claim 1, characterized in that Step (3) Cultivate for 48 to 72 hours.

8. The method according to claim 1, characterized in that: The nucleotide sequence of the Japanese encephalitis virus P3 strain in step (2) is shown in Genebank U47032.

1.

9. A cell line expressing Japanese encephalitis virus prepared by the method according to any one of claims 1 to 8.

10. Use of the method according to any one of claims 1 to 8 or the cell line according to claim 9 in the preparation of Japanese encephalitis vaccine.

Images