HDAC2 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: 2021-06-01
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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Problems solved by technology

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

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  • HDAC2 gene knockout BHK-21 cell line as well as construction method and application thereof
  • HDAC2 gene knockout BHK-21 cell line as well as construction method and application thereof
  • HDAC2 gene knockout BHK-21 cell line as well as construction method and application thereof

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Embodiment 1

[0035]According to the HDAC2 gene sequence of the Mesocricetus Auratus, the GRNA sequence is designed with Crispor software (http: / / crispor.tefor.net / ) of HDAC2: atggcgtacagtcaaggaggg. The design of GRNA was in accordance with the method of Zhang Feng laboratory (NatureProtocols, 2013), synthesized, annealing and attached to PX459 (AddGene # 62988) plasmid. Sequencing the correct CRISPR plasmid. According to the standard transfection procedure of Invitrogen Lipofectamine 2000, CrisPR plasmid transfected with BHK-21 cell lines, and the final concentration of 3 μg / ml was added after 5-7 days after transfection of 48h, and the cell count was carried out, and 100 and 300 respectively. A cell, a single cell clone was formed after a week after formation of cloning cyclones, transferred to 24-well plates, and expanded. Different cell clones were collected in the DNA level and protein levels, respectively. DNA level identification The following primer was amplified by the following primer...

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Abstract

The invention discloses a construction method of an HDAC2 gene knockout BHK-21 cell line. The construction method comprises the following steps: carrying out gene knockout on HDAC2 in a cell line BHK-21 for foot-and-mouth disease vaccine production by utilizing a CRISPR/Cas9 technology; the inventor transfects BHK-21 cells with CRISPR plasmids for knocking out HDAC2, and then separates a plurality of cell clones by using a cloning ring method by combining antibiotic screening with gradient dilution; after genome DNA is extracted, PCR amplification and sequencing are carried out, and cell cloning of which one HDAC2 gene is subjected to homozygous frameshift mutation is successfully recognized; wherein the HDAC2-KO-B3 has the deletion of one basic group at the predetermined cutting position of the Cas9. In the BHK-21 cell line with HDAC2 knockout, the replication rate of foot-and-mouth disease virus is obviously accelerated, the final virus titer is obviously improved, and the cell growth rate is not obviously influenced after HDAC2 knockout, so that the HDAC2 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 HDAC2 from suspension culture type BHK-21 cells and directly applying HDAC2 to production of foot-and-mouth disease vaccines.

Description

Technical field[0001]The present invention belongs to the field of gene engineering, and in particular, the BHK-21 cell line and its construction method and application of HDAC2 gene knockout.Background technique[0002]Foot-Mouth Disease (FMD) is an acute, thermal, highly exposed to infectious diseases caused by foot-and-mouthdisease, FMDV, mainly infected pigs, cattle, sheep, etc. Eashaish animal. There are many ways to spread, and the speed of communication is fast. It has caused huge economic losses around the world, and is listed by the World Animal Health Organization as the first of A-Class A animal disease. At present, the prevention and control of the disease is mainly based on vaccine immunization, and traditional inactivated vaccines still occupy market dominance. The production of the hoof epidemic vaccine is completely dependent on the double-shoe disease virus in BHK-21 (Baby Hamster Kidney Cell) of the pertocrutic kidney cells. BHK-21 cells were earlier by Macpherson an...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12N15/55C12N15/85C12Q1/02C12N7/00
CPCC12N5/0686C12N15/85C12N9/80C12Y305/01098G01N33/5005C12N7/00C12N2310/20C12N2770/32151Y02A40/70
Inventor 孙跃峰龚青侯石桐殷相平王相伟毛箬青
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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