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Method for cultivating hepatitis E viruses

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, can solve the problems of restricted replication of HEV virus, and achieve the effect of promoting replication

Inactive Publication Date: 2016-06-15
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 virus replication is still limited and its culture conditions must be optimized

Method used

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  • Method for cultivating hepatitis E viruses
  • Method for cultivating hepatitis E viruses
  • Method for cultivating hepatitis E viruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Embodiment 1: A549 cell culture

[0020] 1. Take out A549 cells (purchased from ATCC in the United States, number CCL-185) from liquid nitrogen and quickly put them into warm water at 37°C to melt the cell suspension quickly; then add DMEM containing 10% newborn bovine serum (purchased from GIBCO Invitrogen Corporation ) culture solution, adjust the pH to 7.2 and incubate at 37°C for about 4 hours, discard all the culture solution, add fresh culture solution to continue the culture, and after growing into a dense monolayer, wash the cells with PBS (purchased from GIBCO Invitrogen Corporation), trypsin – EDTA (purchased from GIBCO Invitrogen Corporation) digested, added to the above culture medium, placed at 37 ° C, 5% CO 2 Continue culturing in the incubator, such as figure 1 A Normal A549 cells are shown.

[0021] 2. Culture HEV with A549 cells transfected with miRNA-A6

[0022] (1) Prepare a 10% weight-volume ratio of PBS (pH7.4) feces suspension from the collected...

Embodiment 2

[0027] (1) After the genotype IV human HEV virus solution was sterilized by filtration through a 0.22 μm filter membrane, a double antibody solution with 1% volume of the virus solution was added, treated at 4°C for 1 hour, and stored at -80°C for later use, and the virus was detected by Real-timeqPCR The copy number is 2×10 6 Copy number / mL, where the double antibody solution is a solution containing 400U / mL penicillin and 1000U / mL streptomycin;

[0028] (2) Divide A549 cells into 2×10 5 Inoculate each well into a 6-well plate with DMEM medium containing 10% fetal bovine serum at 37°C, 5% CO 2 Cells were cultured statically in an incubator until the cell confluence was 75%-80%; cells were washed 3 times with 1×PBS, serum-free DMEM medium was added, and the miRNA-A6 liposome transfection mixture was dropped into the well plate (miRNA -A6 liposome transfection mixture is prepared by mixing conventional lipofectin transfection reagent and miRNA-A6 sequence at a volume-to-mass ...

Embodiment 3

[0034] (1) After filtering the HEV virus solution through a 0.22 μm filter membrane, add a double antibody solution with a volume of 1.5% of the virus solution, treat it at 4°C for 1 hour, and store it at -80°C for later use. The virus copy number was determined to be 2 by Real-timeqPCR ×10 5 Copy number / mL, where the double antibody solution is a solution containing 400U / mL penicillin and 1000U / mL streptomycin;

[0035] (2) Divide A549 cells into 2×10 5 Inoculate each well into a 6-well plate with DMEM medium containing 10% fetal bovine serum at 37°C, 5% CO 2 Cells were cultured statically in an incubator until the cell confluence was 72%-77%; cells were washed 3 times with 1×PBS, serum-free DMEM medium was added, and the miRNA-A6 liposome transfection mixture was dropped into the well plate (miRNA -A6 liposome transfection mixture is prepared by mixing conventional lipofectin transfection reagent and miRNA-A6 sequence at a volume-mass ratio of 1:4, wherein the miRNA-A6 seq...

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Abstract

The invention discloses a method for cultivating hepatitis E viruses in an in-vitro manner. The method has the advantages that miRNA-A6 [micro-RNA (ribonucleic acid)-A6] is transfected by cells for 24 h, then viruses are inoculated and can be massively replicated, and mass replication of offspring viruses of the HEV (hepatitis E viruses) can be promoted by the transfected miRNA-A6 as revealed by means of Real-Time qPCR (quantitative polymerase chain reaction) and Western Blot analysis; the method is major breakthrough in the aspect of HEV in-vitro cultivation, and excellent foundation is laid for further researching biological characteristics and immunological characteristics of the HEV, researching and developing HEV vaccine and screening anti-HEV medicines.

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 (HEV) is a viral hepatitis pathogen that spreads through the intestinal tract and seriously endangers human health, and can infect humans and various animals across species. HEV is a non-encapsulated single-stranded positive-sense RNA virus, spherical in shape, 27-34nm in diameter and non-encapsulated. The nucleocapsid is icosahedral in three-dimensional symmetry, with protrusions and nicks on the surface, and uneven internal density. The total length of the genome is about 7.3 kb, 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 tr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00C12R1/93
CPCC12N7/00C12N2770/28151
Inventor 黄芬杨臣臣姜典财毕艳红龙飞燕王珏李云龙井申荣
Owner KUNMING UNIV OF SCI & TECH
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