Novel preparation method for hepatitis A virus antigen
A hepatitis A virus and antigen technology, which is applied in the field of preparation of new hepatitis A virus antigens, can solve the problems of failing to take advantage of the advantages of virus culture harvest and medium replacement, high cost of virus culture maintenance medium, and slow proliferation of virus species , to achieve the effect of speeding up the cultivation progress, reducing production costs, and reducing the risk of pollution
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[0033] Such as figure 1 Shown, the present invention provides a kind of novel hepatitis A virus antigen preparation method, it is characterized in that, comprises the steps:
[0034] Step 1. The HAV contained in the culture supernatant of the hepatitis A virus seed L-A-1 attenuated strain with the preservation number CCTCC No.V92004 is cloned, purified and screened to obtain the L-A-1M strain;
[0035] Step 2. Inoculate the L-A-1M strain into human embryonic lung diploid cells, culture in the culture medium, change the maintenance solution once every 10-17 days, collect the supernatant, change the maintenance solution, and take samples, and collect Culture supernatant 5 times;
[0036] Step 3, after the fifth supernatant was collected, digested with trypsin, and collected the virus harvest containing hepatitis A virus with 1 / 5 Hank's solution of the maintenance liquid volume;
[0037] Step 4: Process the harvested virus to obtain the hepatitis A virus antigen.
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[0039] 1. Cells and viruses
[0040] Human embryonic lung diploid cells (2BS strain) and hepatitis A virus L-A-1M strain were provided by Vaccine II Division of Changchun Institute of Biological Products Co., Ltd.
[0041] Human embryonic lung diploid cells (2BS strain) come from the Beijing Institute of Biological Products; the cell bank is under secondary management, namely the master cell bank and the working cell bank; The bank is the 18th passage, and the working cell bank is the 24th passage.
[0042] The preparation process of the hepatitis A virus L-A-1M strain comprises: first, the HAV contained in the culture supernatant of the hepatitis A virus seed L-A-1 attenuated strain is subjected to 10 4 、10 5 、10 6 、10 7 Dilute and add to 2BS cells that have grown into a monolayer in a 24-well plate for culture, add samples to 5 wells for each dilution, collect the liquid every two weeks, add maintenance liquid and detect the antigen content of the collected liquid; secon...
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