HDAC8 gene knockout BHK-21 cell line as well as construction method and application thereof

Abstract

The invention discloses a construction method of an HDAC8 gene knockout BHK-21 cell line, which comprises the following steps: carrying out gene knockout on HDAC8 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 HDAC8, and performing separation to obtain a plurality of cell clones by utilizing antibiotic screening in combination with gradient dilution and a cloning ring method. After genome DNA is extracted, PCR amplification and sequencing are carried out, and cell clones of which two HDAC8 genes are subjected to homozygous frameshift mutation are successfully identified. In the HDAC8 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 HDAC8 knockout has no obvious influence on the cell growth rate, which shows that the HDAC8 knockout BHK-21 cell line has the prospect of being used for foot-and-mouth disease vaccine production. A foundation is laid for further knocking out HDAC8 from suspension culture type BHK-21 cells and directly applying HDAC8 to production of foot-and-mouth disease vaccines.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and particularly relates to a BHK-21 cell line with HDAC8 gene knockout and a construction method and application thereof. 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. cloven hoofed animal. The disease has many routes of transmission and rapid spread, and has caused huge economic losses around the world. At present, the prevention and control of the disease is mainly based on vaccine immunization, and traditional inactivated vaccines still dominate the market. The production of inactivated foot-and-mouth disease vaccine is completely dependent on the replication of foot-and-mouth disease virus in BHK-21 (Baby hamster kidney cell) cells. BHK-21 cells were first established by MacPherson and Stoker in 1962 using 1-day-old Syrian hamst...

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However, the role of protein acetylation and HDACs family genes in

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