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

A BHK-21, gene knockout technology, applied in the field of genetic engineering, can solve the problem of unclear effect, and achieve the effect of accelerating the replication rate and increasing the virus titer

Active Publication Date: 2021-06-18
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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

Method used

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

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Embodiment 1

[0034] According to the HDAC8 gene sequence of golden hamster (Mesocricetus auratus) in NCBI, the gRNA sequence was designed in the first exon region of HDAC8 using CRISPOR software (http: / / crispor.tefor.net / ): GCTCTTCTGATCGGCCCGGG. The designed gRNA was synthesized, annealed and ligated to the PX459 (Addgene#62988) plasmid according to the published method of Zhang Feng's laboratory (Nature Protocols, 2013). The correctly sequenced CRISPR plasmids were extracted and used. According to the standard transfection procedure of Invitrogen Lipofectamine 2000, CRISPR plasmid was transfected into BHK-21 cell line. After 48 hours of transfection, puromycin with a final concentration of 3 μg / mL was added for 5-7 days, and the cells were counted and plated with 100 cells and 300 cells After a week, single cell clones were formed and single clones were picked with a cloning ring, transferred to 24-well plates, and expanded for culture. Different cell clones were collected for identifica...

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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...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/55C12N5/10C12N7/00C12Q1/70C12Q1/6851C12Q1/02C12Q1/06G01N33/569C12R1/91C12R1/93
CPCC12N15/85C12N9/22C12N9/80C12N5/0686C12N7/00C12Q1/701C12Q1/6851C12Q1/02G01N33/56983G01N33/48735C12Y305/01098C12N2800/107C12N2510/00C12N2770/32151G01N2333/09G01N2469/10C12Q2531/113C12Q2545/101C12Q2563/107Y02A40/70
Inventor 孙跃峰侯石桐毛箬青张会军殷相平赵帅阳
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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