HDAC3 gene knockout BHK-21 cell line as well as construction method and application thereof

Abstract

The invention discloses a construction method of an HDAC3 gene knockout BHK-21 cell line, which comprises the following steps: carrying out gene knockout on HDAC3 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 HDAC3, 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 clones of which two HDAC3 genes are subjected to homozygous frameshift mutation. In the HDAC3 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 HDAC3 knockout has no obvious influence on the cell growth rate, so that the HDAC3 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 HDAC3 from suspension culture type BHK-21 cells and directly applying HDAC3 to foot-and-mouth disease vaccine production.

Description

technical field

[0001] The invention belongs to the field of genetic engineering, and in particular relates to a BHK-21 cell line with HDAC3 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...

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