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

Pending Publication Date: 2020-01-10
长春生物制品研究所有限责任公司
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Problems solved by technology

[0008] Based on the problems in the prior art, the present invention has designed and developed a novel hepatitis A virus antigen preparation method. The purpose of the present invention is to solve the slow proliferation of most of the used hepatitis A virus species and the high cost of virus culture and maintenance solution. , the amount of virus harvested is small, and the advantages of continuous multiple harvests and medium changes of virus culture cannot be brought into play

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  • Novel preparation method for hepatitis A virus antigen
  • Novel preparation method for hepatitis A virus antigen
  • Novel preparation method for hepatitis A virus antigen

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preparation example Construction

[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.

Embodiment

[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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Abstract

The invention discloses a novel preparation method for a hepatitis A virus antigen. The preparation method comprises the following steps of step one, performing cloning, purification and screening onHAV contained in a cultural supernatant of a hepatitis A virus strain L-A-1 with a preservation number being CCTCC No. V92004 to obtain an L-A-1M strain; step two, inoculating the L-A-1M strain to human embryonic lung diploid cells, performing culturing in a culture solution, replacing a maintenance solution every 10-17 days, collecting a supernatant, replacing the maintenance solution, preservinga sample, and totally collecting cultural supernatants for 5 times; step three, after collecting the fifth supernatant, collecting a hepatitis A virus contained virus harvest by a Hank's solution with the quantity being 1 / 5 of the quantity of the maintenance solution after enzymic digestion with trypsin; and step four, treating the virus harvest to obtain the hepatitis A virus antigen.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for preparing a novel hepatitis A virus antigen. Background technique [0002] Hepatitis A is an acute and usually self-limiting infectious disease caused by hepatitis A virus (HAV). HAV is transmitted through the intestinal tract and is mainly transmitted in the population through the fecal-oral route. A large number of practices have shown that hepatitis A vaccination is the most effective measure to prevent and control the prevalence of hepatitis A. [0003] Hepatitis A virus (HAV) is a non-enveloped small RNA virus, which was named enterovirus 72 in 1982, but further research found that it has at least 6 differences with other small RNA viruses (enteroviruses) Differences: (1) The nucleotide and amino acid sequence characteristics of HAV are different from those of other viruses; (2) HAV adapts, grows, and replicates slowly in cell culture; HAV has a long replication cy...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00C07K14/10C12R1/93
CPCC12N7/00C07K14/005C12N2770/32451
Inventor 岳立广徐艳玲刘令九夏青娟王艺博张宏丽任琦付艳杰李丽莉王春雨
Owner 长春生物制品研究所有限责任公司
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