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Method for cultivating hepatitis e virus

A technology of hepatitis E virus and virus, which is applied in the field of successfully culturing hepatitis E virus strains in vitro and improving the replication efficiency of progeny viruses, and can solve problems such as restricted replication of HEV viruses

Inactive Publication Date: 2015-11-18
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, HEV viral replication is still limited, necessitating optimization of

Method used

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  • Method for cultivating hepatitis e virus
  • Method for cultivating hepatitis e virus
  • Method for cultivating hepatitis e virus

Examples

Experimental program
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Effect test

Embodiment 1

[0019] Embodiment 1: A549 cell culture

[0020] A549 cells (purchased from ATCC, USA, number CCL-185) were taken out of liquid nitrogen and quickly placed in warm water at 37°C to rapidly melt the cell suspension; then cultured in DMEM containing 10% newborn bovine serum (purchased from GIBCO Invitrogen Corporation) Adjust the pH to 7.2 and incubate at 37°C for about 4 hours. Discard all the culture medium and add fresh culture medium to continue the culture. After growing into a dense monolayer, wash the cells with PBS (purchased from GIBCO Invitrogen Corporation) and trypsin-EDTA (purchased from GIBCO Invitrogen Corporation) to digest, add the above culture medium, and place at 37°C, 5% CO 2 Continue culturing in the incubator, such as figure 1 A Normal A549 cells are shown.

Embodiment 2

[0021] Embodiment 2: Cultivate HEV with the DMEM medium that adds the pregnant woman's late serum

[0022] 1. Prepare a PBS (pH7.4) feces suspension with a weight-volume ratio of 10% from the collected HEV-positive feces for HEV isolation, shake vigorously to emulsify the feces, centrifuge at 12,000g for 10 minutes at 4°C, and collect the supernatant. 0.22μm filter membrane filter to sterilize, add double antibody solution (400U / mL penicillin and 1000U / mL streptomycin) with 2% volume of virus solution, treat at 4°C for 1h, store at -80°C for later use, and detect the virus by Real-timeqPCR The copy number is 2×10 6 Copy number / mL;

[0023] Isolated from HEVRNA-positive pig feces samples in Kunming, Yunnan, China, the genotype is type 4, and the GenBank database number is No.JF747598;

[0024] 2. Dilute the cells (A549 cells) in Example 1 by 2×10 5 Inoculate each well into a 6-well plate, and use DMEM medium containing 10% fetal bovine serum by mass percentage at 37 °C, 5% C...

Embodiment 3

[0033] Example 3: (1) HEV virus (isolated from HEVRNA-positive pig feces samples in Kunming, Yunnan, China, genotype 4, GenBank database number No. JF747598) solution was sterilized through a 0.22 μm filter membrane, and the virus was added Double antibody solution with 1% solution volume, treated at 4°C for 1 hour, and stored at -80°C for later use, the virus copy number was determined to be 2×10 by Real-timeqPCR 6 Copy number / mL, where the double antibody solution is a solution containing 400U / mL penicillin and 1000U / mL streptomycin;

[0034] (2) Divide A549 cells into 2×10 5 Inoculate each well into a 6-well plate, and use DMEM medium containing 10% fetal bovine serum by mass percentage at 37 °C, 5% CO 2 Culture the cells statically in the incubator until the cells grow into a monolayer;

[0035] (3) Inoculate 100 μL of the HEV virus suspension in step (2) into a single layer of cells, incubate at 37°C for 1 hour, and shake gently every 15 minutes to make the virus fully ...

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Abstract

The invention discloses a method for cultivating hepatitis e virus. The method comprises the steps that a serum of a later pregnant woman is added into an HEB strain obtained through separation of an epidemic disease investigation for in-vitro culture, the virus can be replicated massively, and immunofluorescence and Western Blot analysis are utilized for revealing that the serum of the later pregnant woman can promote massive replication of HEV progeny virus. According to the method, great breakthrough is made in the aspect of HEV in-vitro culture, and a good foundation is laid for further researching the biological and immunological characteristics of HEV and the HEV infection and pathogenic mechanism and screening and identifying anti-HEV drugs.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a new method for successfully cultivating hepatitis E virus strains in vitro and improving the replication efficiency of progeny viruses. Background technique [0002] Hepatitis E virus (Hepatitis EVirus, HEV) is a viral hepatitis pathogen transmitted through the intestinal tract, which can infect humans and various animals across species. HEV is a non-enveloped single-stranded positive-sense RNA virus. The size of spherical virus particles is 27-34nm. There are protrusions and nicks on the surface, and the internal density is uneven. The total length of the genome is about 7.3kb, consisting of 3 open reading frames. There are mainly four genotypes of HEV in the world, and type IV HEV strains are common in China. The transmission route of HEV is mainly through the fecal-oral route, but can also be transmitted through other routes, including blood transmission, contact transmission, ve...

Claims

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Application Information

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IPC IPC(8): C12N7/00
Inventor 黄芬毕艳红杨臣臣赵显晨马天武井申荣
Owner KUNMING UNIV OF SCI & TECH
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