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A kind of method for cultivating hepatitis E virus in vitro

A hepatitis E virus, in vitro culture technology, applied in the directions of viruses, viruses/phages, biochemical equipment and methods, etc., can solve the problem that cannot use cancer cell line HEV vaccine research and development and production, cannot be stably continuously subcultured, cannot be real. Reaction-related symptoms, etc.

Active Publication Date: 2021-10-26
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

On the one hand, the cell lines used in the currently established cell culture system are all cancer cell lines, which cannot truly reflect the related symptoms of normal organisms infected with HEV, let alone use cancer cell lines for the development and production of HEV vaccines
On the other hand, when using the existing cell culture system, it is necessary to inoculate a higher titer of virus (10 8 copy / mL) to maintain culture in vitro, and the virus titer will decrease in offspring cell suspension, which cannot be stably and continuously subcultured

Method used

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  • A kind of method for cultivating hepatitis E virus in vitro
  • A kind of method for cultivating hepatitis E virus in vitro
  • A kind of method for cultivating hepatitis E virus in vitro

Examples

Experimental program
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Effect test

Embodiment 1

[0024] Embodiment 1: Hepatitis E virus cultured in vitro by MDCK cells

[0025] 1. Take out MDCK cells (purchased from ATCC in the United States) from liquid nitrogen and immediately put them into warm water at 37°C to melt the cell suspension quickly; centrifuge at 1500 rpm for 5 minutes, then transfer to 10cm 3 Add DMEM culture solution (purchased from GIBCO Invitrogen Corporation) containing 10% newborn bovine serum by volume to the cell culture flask, culture at 37°C, and after growing into a dense monolayer, wash with PBS (purchased from GIBCO Invitrogen Corporation) Wash the cells, digest with trypsin-EDTA (purchased from GIBCO Invitrogen Corporation), add the above culture medium, and place at 37°C, 5% CO 2 Continue culturing in the incubator;

[0026] 2. Prepare a 10% weight-volume ratio of PBS (pH7.4) feces suspension 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 colle...

Embodiment 2

[0028] Embodiment 2: Continuous culture of hepatitis E virus in MDCK cells in vitro

[0029] 1. Take out MDCK cells (purchased from ATCC in the United States) from liquid nitrogen and immediately put them in warm water at 37°C to melt the cell suspension quickly; centrifuge at 1500 rpm for 5 minutes, then transfer to 10cm 3Add DMEM (purchased from GIBCO Invitrogen Corporation) culture solution containing 10% newborn bovine serum (purchased from GIBCO Invitrogen Corporation) to the cell culture flask, and culture at 37°C. Wash the cells, digest with trypsin-EDTA (purchased from GIBCO Invitrogen Corporation), add the above culture medium, and place at 37°C, 5% CO 2 Continue culturing in the incubator;

[0030] 2. Prepare a PBS (pH7.4) feces suspension with a weight-volume ratio of 15% 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. Filter and sterilize thr...

Embodiment 3

[0034] Example 3: Observation of HEV-infected MDCK cells by fluorescence confocal microscope

[0035] 1. Take out MDCK cells (purchased from ATCC, USA) from liquid nitrogen and immediately put them into warm water at 37°C to melt the cell suspension quickly; centrifuge at 1500 rpm for 5 minutes, then transfer to 10cm 3 Add DMEM (purchased from GIBCO Invitrogen Corporation) culture solution containing 10% newborn bovine serum by volume to the cell culture flask, culture at 37°C, and wash with PBS (purchased from GIBCO Invitrogen Corporation) after growing into a dense monolayer Cells were digested with trypsin-EDTA (purchased from GIBCO Invitrogen Corporation), added to the above culture medium, and placed at 37°C, 5% CO 2 Continue culturing in the incubator;

[0036] 2. Prepare a PBS (pH7.4) feces suspension with a weight-volume ratio of 15% from the collected HEV-positive feces for HEV isolation, shake vigorously to emulsify the feces, centrifuge at 12,000g for 10 minutes at...

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Abstract

The invention discloses a method for culturing hepatitis E virus in vitro. The method uses MDCK cells to culture hepatitis E virus in vitro, and uses RT-nPCR, PT-qPCR and immune confocal methods to confirm that the hepatitis E virus can MDCK cells are replicated in large quantities, and can be stably passed down for more than 10 generations. This method has made a major breakthrough in the in vitro culture of hepatitis E virus. In order to further study the biological and immunological characteristics of hepatitis E virus, HEV infection and pathogenicity The mechanism has laid a good foundation.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to a new method for successfully culturing and continuously replicating hepatitis E virus in vitro. Background technique [0002] Hepatitis E Virus (HEV) is a viral hepatitis pathogen transmitted through the intestinal tract, which can infect humans and various animals across species. HEV is a single-stranded positive-sense RNA virus. Current studies have found that there are mainly 8 genotypes (HEV-1---HEV-8), and the genotype 4 HEV strain (HEV-4) is mainly prevalent in China. It has been confirmed that the transmission route of HEV is mainly the fecal-oral route, and it can also be transmitted by other routes, such as blood transmission, contact transmission, vertical transmission, and organ transplantation. [0003] HEV infection is generally an acute self-limiting infection, and patients can recover after 4-6 weeks of self-detoxification. However, immunodeficiency patients and the el...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/00
CPCC12N7/00C12N2770/28151
Inventor 黄芬郝先辉禹文海
Owner KUNMING UNIV OF SCI & TECH
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