A kind of culture method of hepatitis E virus and preparation method of inactivated vaccine
A technology for hepatitis E virus and hepatitis E, which is applied in the field of culture methods and the preparation of inactivated vaccines, and can solve the problems of no vaccine sales, and the protection properties need to be further evaluated.
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
preparation example Construction
[0034] In the preparation method of hepatitis E inactivated vaccine of the present invention:
[0035] Step (a) The cell matrix is Vero cells, the cell culture medium is DMEM medium containing 10% fetal bovine serum, and the culture temperature is usually 35.5-37.5°C.
[0036] The medium in step (b) is DMEM medium containing 2% fetal bovine serum, and the culture temperature is usually 35.5-37.5°C.
[0037] The preparation of the vaccine from the purified virus in step (d) can be carried out using conventional methods in the art, for example, the following method can be used: first, the cells selected in step (b) are repeatedly frozen and thawed at -60°C to -80°C for 3-5 times, Obtain virus-cell culture suspension. Then centrifuge at 8000rmp for 30-50min, and collect the supernatant. Concentrate with 10% PEG (molecular weight 6,000) and 0.4M NaCl solution, centrifuge at 10,000 rpm for 50-80 min, discard the supernatant, and collect the precipitate. After suspending the co...
Embodiment 1
[0039] Embodiment 1: the culture method of hepatitis E virus strain KM01
[0040] (a) The virus was isolated from feces of pigs acutely infected with hepatitis E, and was confirmed to be hepatitis E virus through identification of biological characteristics.
[0041] The specific separation method is:
[0042] Take feces from pigs acutely infected with hepatitis E in a 50ml centrifuge tube, add DEPC water and stir at 12000rmp, centrifuge at 4°C for 15min, take the supernatant and filter it through 0.45um and 0.22um microporous filters respectively, and then contain 400U / mL penicillin at a ratio of 1:1000 and 1000U / mL streptomycin double antibody solution, and treated at 4°C for 2 hours to obtain a virus suspension, which was confirmed to be hepatitis E virus by biological characteristics identification.
[0043] (b) Blind passage on Vero cells was adapted for 6 passages and still had a high virus titer (>10 7 ).
[0044] (c) Gradual reduction of culture time during culture ad...
Embodiment 2
[0048] Embodiment 2: the preparation of hepatitis E inactivated vaccine
[0049] Take out the Vero cells from the liquid nitrogen container, centrifuge at 1000rmp for 10min, discard the original preservation solution, add DMEM medium containing 10% fetal bovine serum, culture at 37°C until the cells grow into a monolayer, wash with PBS, trypsinize, Add the above-mentioned medium and continue to subculture until enough cells are obtained.
[0050] When the above cells were cultured at a concentration of 106 / ml, the Hepatitis E virus strain KM01 obtained in Example 1 was added to the pH 7 medium, and cultured at 37° C. for 7 days. After the culture period expired, the cells were repeatedly frozen and thawed three times at -80°C to obtain a virus-cell culture suspension. Then centrifuge at 8000rmp for 30min, and collect the supernatant. Concentrate with 10% PEG (molecular weight 6000) and 0.4M NaCl solution, centrifuge at 10000 rpm for 60 min, discard the supernatant, and colle...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More