Hepatitis A virus strain, method for preparing hepatitis A inactivated vaccine and obtained vaccine
A hepatitis A virus and hepatitis A technology, applied in biochemical equipment and methods, virus/bacteriophage, and vector-borne diseases, can solve the problems of high manufacturing cost and low reproduction efficiency of hepatitis A inactivated vaccines, and achieve Guaranteed effectiveness, high virus yield, and reduced production costs
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preparation example Construction
[0028] In the method for preparing the hepatitis A inactivated vaccine of the present invention, the cell matrix described in step (a) is Vero cell, and this cell can be purchased from ATCC, for example, and culture medium is the MEM that has serum culture medium such as containing 10% serum As for the culture medium, the culture temperature is well known to those skilled in the art, usually 36.5-37.5°C.
[0029] The culture medium in step (b) is a serum-containing culture medium such as MEM culture medium containing 2% serum, and the culture temperature is well known to those skilled in the art, usually 34.5-35.5°C, and no cytopathic pathology (CPE) occurs during the culture process.
[0030] 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 virus-cell culture suspension is ultrasonically crushed until the cell disruption rate reaches more than 9...
Embodiment 1
[0031] The present invention is described in more detail with examples below. Embodiment 1: the breeding method of hepatitis A virus strain YN5
[0032] (a) The virus was isolated from the feces of clinical hepatitis A patients, and tested by biological characteristics
[0033] Confirmed to be hepatitis A virus;
[0034] (b) Blind on diploid cell 2BS (source: China Institute for the Control of Pharmaceutical and Biological Products)
[0035] Adapted to 6 generations;
[0036] (c) Then cultivate and adapt to Vero for 40 days, and then gradually reduce the culture time until
[0037] To the peak of proliferation at 14 days.
[0038] Wherein, after each culture period expires, the virus-cell culture suspension is obtained by digestion with trypsin-EDTA, and stored at -60°C to -80°C for future use. Before inoculation, the cells were crushed by ultrasonic waves until the cell crushing rate reached over 95%. Vaccinations were performed at an infectious dose (M.O.I.) o...
Embodiment 2
[0039] Finally, a desired Hepatitis A virus strain with desired characteristics was obtained, named YN5, which was deposited in China Center for Type Culture Collection on April 20, 2001, with the preservation number CCTCC NO: V200104. Embodiment 2: the preparation of hepatitis A inactivated vaccine
[0040] Take out the Vero cell ampoule (source: National Institute for the Control of Pharmaceutical and Biological Products) from the liquid nitrogen container, centrifuge at 1000rpm for 10 minutes, discard the original preservation solution, add the MEM nutrient solution containing 10% neonatal bovine serum, 0.20% hydrolyzed milk protein (with 7% NaHCO 3 Adjust the pH to 6.8) and culture at 37°C. After the cells grow into a single layer, they are washed with PBS, digested with trypsin-EDTA, added to the above nutrient solution, and subcultured once every 5 days at a seeding rate of 1:2 until the cells are obtained. Sufficient cells for bulk production.
[0041] The above cells...
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