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Preparation method and application of anti-red-spot grouper neuronecrosis virus (tcm) embryonic stem cells

An embryonic stem cell and red-spotted grouper technology is applied in the field of preparation of medaka haploid embryonic stem cells resistant to fish nerve necrosis virus, which can solve the economic loss of the world aquaculture industry, the unclear pathogenic mechanism, and the inconsistency of host genes. question of clarity

Active Publication Date: 2019-02-01
SUN YAT SEN UNIV
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Problems solved by technology

The fish neuronecrosis caused by this virus is a worldwide popular fish infectious disease. So far, more than 120 kinds of fish have been infected by this virus, and the mortality rate of infected fish is as high as 50-100%. The world's aquaculture industry causes huge economic losses
However, the pathogenic mechanism of the virus is still unclear, especially the host genes directly related to the virus infection are still unclear

Method used

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  • Preparation method and application of anti-red-spot grouper neuronecrosis virus (tcm) embryonic stem cells
  • Preparation method and application of anti-red-spot grouper neuronecrosis virus (tcm) embryonic stem cells
  • Preparation method and application of anti-red-spot grouper neuronecrosis virus (tcm) embryonic stem cells

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Embodiment 1

[0043] In this embodiment, RGNNV comes from this laboratory; ENU (Sigma) is dissolved in equal volumes of ethanol and PCS buffer (50mM citric acid, 100mM sodium hydrogen phosphate, pH=5.0) with a mass fraction of 95%; O 6 -BG dissolved in dimethyl sulfoxide; ESM4 medium is the fourth ES cell culture medium for medaka, based on DMEM (Dulbecco's Modified Eagle Medium), supplemented with growth factors, medaka embryo extract and fish Serum is formulated.

[0044] (1) Cultivate HX1 cells in ESM4 medium, add MEE, fish serum and bFGF as protein supplements, digest the cells with 0.25% trypsin and transfer them to 6-well cell culture plates covered with gelatin, 10 5 cells / well, placed in a 28°C incubator for 24 hours;

[0045] (2) ENU mutagenesis: adding O at a concentration of 10 μM in the medium 6 -BG pretreatment for 24 hours, then remove the medium, replace it with ESM4 medium with a final concentration of ENU of 10 mg / ml, continue to cultivate for 48 hours, about 50-60% of th...

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Abstract

The invention belongs to the technical field of biology, and particularly relates to a preparation method and application of an anti-Epinephelus akaara nervous necrosis virus medaka haploid embryonicstem cell. Medaka haploid embryonic stem cells (HX1 cells) are induced by Ethylmtrosourea (ENU)), and then the anti-Epinephelus akaara nervous necrosis virus medaka haploid embryonic stem cell G1 canbe screened; the anti-Epinephelus akaara nervous necrosis virus medaka haploid embryonic stem cell G1is preserved in china center for type culture collection on January 17 th, 2018, the preservation address is wuhan university in china, and the preservation number is CCTCC NO: C201832. The anti-Epinephelus akaara nervous necrosis virus medaka haploid embryonic stem cell G1 provides an experiment basis and a theoretical basis for further identifying host factors participating in virus infection, and provides a new way for genetic breeding of antiviral fishes.

Description

technical field [0001] The invention belongs to the field of biological technology, and in particular relates to a preparation method and application of medaka haploid embryonic stem cells resistant to fish nerve necrosis virus. Background technique [0002] Haploid cells have only one set of chromosomes, no alleles, each gene in the cell has only one, and each gene can play its own role, so haploid cells are good materials for studying gene functions. At present, haploid cells have been widely used in gene screening and identification, etiology, animal breeding and transgenic animals. For example, by performing deep gene insertion mutations on human haploid cells, it was successfully identified that mucopolysaccharides are closely related to Rift Valley fever virus infection. Through genome-wide loss-of-function screening experiments on human haploid cells, a series of host factors associated with hantavirus membrane fusion were successfully identified. [0003] Haploid e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/01C12N5/0735
CPCC12N5/0606C12N15/01
Inventor 贾坤同易梅生张湾湾
Owner SUN YAT SEN UNIV
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