HDAC5 gene knockout BHK-21 cell line as well as construction method and application thereof

Abstract

The invention discloses a construction method of an HDAC5 gene knockout BHK-21 cell line, which comprises the following steps: carrying out gene knockout on HDAC5 in a cell line BHK-21 for foot-and-mouth disease vaccine production by utilizing a CRISPR / Cas9 technology, transfecting BHK-21 cells by utilizing CRISPR plasmids for knocking out the HDAC5, and separating the cells to obtain a plurality of cell clones by utilizing antibiotic screening in combination with gradient dilution and a cloning ring method; extracting the genome DNA, performing PCR amplification and sequencing and successfully identifying the cell cloning of which one HDAC5 gene is subjected to homozygous frameshift mutation, wherein the HDAC5-KO-A2 has deletion of 13 basic groups at a Cas9 predetermined cutting position. In the HDAC5 knockout BHK-21 cell line, the replication rate of foot-and-mouth disease virus is obviously accelerated, the final virus titer is obviously improved, and the HDAC5 knockout has no obvious influence on the cell growth rate, so that the HDAC5 knockout BHK-21 cell line has the prospect of being used for foot-and-mouth disease vaccine production. Therefore, the foundation is laid for further knocking out HDAC5 from suspension culture type BHK-21 cells and directly applying HDAC5 to production of foot-and-mouth disease vaccines.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a BHK-21 cell line with HDAC5 gene knockout and its construction method and application. Background technique [0002] Foot-and-mouth disease (FMD) is an acute, febrile, highly contagious disease caused by foot-and-mouth disease virus (FMDV) infection, mainly infecting pigs, cattle, sheep, etc. Artiodactyl. The disease has many transmission routes and fast transmission speed, and has caused huge economic losses worldwide. It is listed as the first class A animal disease by the World Organization for Animal Health. At present, the prevention and control of the disease is mainly based on vaccine immunization, and traditional inactivated vaccines still occupy a dominant position in the market. The production of inactivated foot-and-mouth disease vaccine completely depends on the replication of foot-and-mouth disease virus in BHK-21 (Baby hamster kidney cell). BHK-21...

AI Technical Summary

Problems solved by technology

However, the role of protein acetylation and HDACs family genes in the process of foot-and-mouth disease virus infection is not clear

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