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Method for knocking out rabies virus genome mediated by CRISPR-Cas9 system

A rabies virus and genome technology, applied in other methods of inserting foreign genetic materials, gene therapy, antiviral agents, etc., to achieve the effect of inhibiting virus invasion

Pending Publication Date: 2019-06-28
INNER MONGOLIA UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

For the knockout of rabies virus gene, there are few reports

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  • Method for knocking out rabies virus genome mediated by CRISPR-Cas9 system
  • Method for knocking out rabies virus genome mediated by CRISPR-Cas9 system
  • Method for knocking out rabies virus genome mediated by CRISPR-Cas9 system

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Embodiment 1

[0036] Example 1 The method for knocking out the rabies virus genome mediated by CRISPR-Cas9 system

[0037] 1. Materials and methods

[0038] 1.1 Test material

[0039] CRISPR-Cas9 plasmid PX459 (Addgene, catalog number: 62988, http: / / www.addgene.org / 62988 / ), lipofectamine TM 3000 was purchased from Invitrogen, Escherichia coli DH5α and stableIII were purchased from Beijing Quanshijin Biotechnology Co., Ltd., BbsI and other restriction enzymes, plasmid mini-extraction kits, DNA extraction kits, and DNA purification kits were purchased from the United States. therom company. Marker, Primer star GXL, and pMD19T were all purchased from Dalian Bao Biological Company. Endotoxin-free plasmid extraction kit was purchased from Promega, DMEM high-glucose medium, Opti-MEM TM Serum-free culture medium, double antibodies, and fetal bovine serum were purchased from Gbico. All other reagents were of domestic analytical grade. The rabies virus strains used were CVS-B2C, SAD, DRV, and...

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Abstract

The invention provides a method for knocking out a rabies virus genome mediated by a CRISPR-Cas9 system. According to gene sequences of rabies virus genes G and N, a gRNA sequence based on the CRISPR-Cas9 system is designed, the designed gRNA sequence is connected into a CRISPR-Cas9 vector, and then the connected vector is transferred into a cell infected with rabies viruses for blocking virus replication. According to the method, an effective solution is provided for the treatment of rabies by constructing the novel CRISPR-Cas9 vector. By constructing the gene-modified CRISPR-Cas9 system, glycoprotein expression is blocked, virus invasion is inhibited, and the elimination of intracellular viruses is achieved. Experiments show that the CRISPR-Cas9 system has a significant inhibitory effecton the rabies viruses.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for knocking out the rabies virus genome mediated by a CRISPR-Cas9 system to inhibit virus replication. Background technique [0002] Rabies is widely distributed all over the world. According to the statistics of the World Health Organization (WHO), more than 55,000 people die from rabies every year in the world, and about 95% of them occur in developing countries in Asia and Africa. It is a zoonotic disease that seriously endangers global public health. suffer from an infectious disease. Rabies virus (Rabies virus RV) is a single-stranded negative-sense RNA virus belonging to the genus Lyssavirus in the family Rhabdoviridae. The particle size of the virus is about 75nm, the length is about 100-300nm, and its shape is a typical bullet. Its genome is a Non-segmented single-stranded negative-strand RNA, about 12000nt, contains 5 structural proteins, one from 3' to 5': nucleo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/63C12N15/90A61K48/00A61P31/14
Inventor 杨洋赵彩权李光鹏刘晓燕张金玲
Owner INNER MONGOLIA UNIVERSITY
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