Breeding of hepatitis A virus Chinese strain and attenuated strain and its complementary DNA sequence

A technology for hepatitis A virus and virus strains, applied in inactivation/attenuation, virus/phage, botanical equipment and methods, etc., can solve the problem that the original strain virus and related strain sequences have not been reported yet.

Inactive Publication Date: 2002-02-27
ZHEJIANG PUKANG BIOTECH
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Problems solved by technology

But so far there is no sequence report of the or

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0033] Isolation of Hepatitis A Virus Gkm Strain and Its cDNA Sequence

[0034] Collect stool samples from patients with hepatitis A in the acute phase in the suburbs of Hangzhou, make a 20% (V / V) suspension with PBS (pH 7.0), extract with F113-PEG precipitation and differential centrifugation, and the extract is precipitated by ultracentrifugation. Hepatitis A virus particles with a size of 27nm were observed by immunoelectron microscopy, which was the preliminary purified HAVGkm strain.

[0035] The virus ribonucleic acid (RNA) of the HAV Gkm strain was extracted by the guanidine salt / hot phenol method, and the cDNA gene library of the HAV Gkm strain was obtained by using a cDNA synthesis kit with high-fidelity polymerase chain reaction (PCR) technology and molecular cloning technology. The cDNA sequence of the HAV Gkm strain was obtained by using the DNA sequence analysis kit and the sequence analysis of the existing cDNA clones by the dideoxy terminal termination method, a...

Embodiment 2

[0037] Isolation of Hepatitis A Virus H2K5 Strain and Its Partial cDNA Sequence

[0038] HAV Gkm strain virus seeds were inoculated with human embryonic lung diploid KMB17 monolayer cells, adsorbed at 35°C for 2 hours, cultured at 35°C with Eagles of newborn bovine serum and milk protein maintenance solution, and changed the maintenance solution once a week until the first day At 6 weeks, remove the original maintenance solution, digest with trypsin-EDTA solution, collect the cells, extract HAV by freezing and thawing, and sonicate, and then use the HAV as the virus seed for the next round of passage. The KMB17 cells were passed continuously for 5 generations in the above method, and the proliferation of the virus was monitored by direct immunofluorescence or ELISA in each generation, so as to obtain the HAVH2K5 strain.

[0039] With reference to the HAV Gkm strain cDNA sequence, synthesize 3 pairs of reverse transcription-polymerase chain reaction (RT-PCR) primers located in ...

Embodiment 3

[0042] Isolation of Hepatitis A Virus H2K20 Strain and Its Partial cDNA Sequence

[0043] HAV Gkm strain virus seeds were inoculated with human embryonic lung diploid KMB17 monolayer cells, adsorbed at 35°C for 2 hours, cultured at 35°C with Eagles of newborn bovine serum and milk protein maintenance solution, and changed the maintenance solution once a week until the first day At 6 weeks, remove the original maintenance solution, digest with trypsin-EDTA solution, collect the cells, extract HAV by freezing and thawing, and sonicate, and then use the HAV as the virus seed for the next round of passage. The KMB17 cells were continuously passaged in the above method, 15 passages at 35°C, 5 passages at 32°C, and the proliferation of the virus was monitored by direct immunofluorescence or ELISA at each passage, thereby obtaining the HAV H2K20 strain.

[0044]Referring to the cDNA sequence of the HAV Gkm strain, synthesize 3 pairs of RT-PCR primers located in the HAV5' NCR region, ...

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Abstract

The present invention relates to hepatitis A virus Chinese strain and attenuated strain separation and cultivation and their complementary DNA sequence, in particular, it relates to separation and purification of hepatitis A virus Chinese strain, cultivation of series attenuated strain and its unique cDNA sequence. Said series cDNA series can provide basic material for further understanding genetic property and genetic stability of hepatitis A virus strain and expounding molecular mechanism of HAV attenuation, and can create condition for developing novel hepatitis A vaccine which can be quickly grown and possesses high titre and high antigenicity and can provide scientific basis for developing gene engineering polypeptide vaccine.

Description

technical field [0001] The invention relates to hepatitis A virus (HAV), especially the complementary deoxyribonucleic acid (cDNA) sequence of the HAV Chinese popular strain Gkm and its attenuated strain, as well as the cultivation method, process and conditions of the attenuated strain. Background technique [0002] Hepatitis A is a worldwide infectious disease, which is caused by hepatitis A virus (HAV), and about 4 billion people in the world are threatened. In China, among adults over 35 years old, the infection rate of hepatitis A reaches 90%, which often causes seasonal epidemics. Due to the high incidence and long course of the disease, it has seriously affected people's health and economic development. According to the actual situation in our country, the effective and fundamental measure to control the prevalence of hepatitis A lies in the development of vaccines, and the key to vaccines is good attenuated strains. [0003] In 1986, Provost ...

Claims

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Application Information

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IPC IPC(8): C12N7/01C12N7/08C12N15/51C12Q1/70
Inventor 毛江森陈勇柴少爱唐彩华庄昉成陈念良黄海鹰钱汶洪艳忻亚娟毛子安谢汝瑛
Owner ZHEJIANG PUKANG BIOTECH
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