Construction method of porcine RIPK3 gene deletion cell strain capable of promoting proliferation of pseudorabies virus as well as product and application of porcine RIPK3 gene deletion cell strain

A pseudorabies virus and gene deletion technology, applied in the biological field, can solve problems such as unclear virus clearance mechanism, and achieve the effect of promoting proliferation

Pending Publication Date: 2022-05-27
INST OF ANIMAL HEALTH GUANGDONG ACADEMY OF AGRI SCI
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Problems solved by technology

At present, the virus clearance mechanism mediated by RIPK3 is not clear, because RIPK3 is closely related to the regulation process of cell metabolism and oxidative stress in addition to participating in programmed cell necrosis.

Method used

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  • Construction method of porcine RIPK3 gene deletion cell strain capable of promoting proliferation of pseudorabies virus as well as product and application of porcine RIPK3 gene deletion cell strain
  • Construction method of porcine RIPK3 gene deletion cell strain capable of promoting proliferation of pseudorabies virus as well as product and application of porcine RIPK3 gene deletion cell strain
  • Construction method of porcine RIPK3 gene deletion cell strain capable of promoting proliferation of pseudorabies virus as well as product and application of porcine RIPK3 gene deletion cell strain

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Embodiment 1

[0033] Cells, viruses and plasmids: Porcine kidney epithelial cells (PK15) were purchased from China Center for Type Culture Collection (Wuhan University), and PRVBartha-K61 vaccine strain (PRVBartha-K61) was purchased from China Veterinary Microorganism Collection Center, PRV GD-WH strain (PRV GD-WH) was isolated and preserved in the Swine Disease Laboratory, Institute of Animal Health, Guangdong Academy of Agricultural Sciences. Escherichia coli Trans10 competent cells were purchased from Complete Gold Company. CRISPR / Cas9 vector plasmid pX459 pSpCas9-2Apuro-MCS and helper vector plasmid EZ-GuideXH were purchased from addgene company.

[0034]Reagents and antibodies: Restriction endonucleases BbsI, HindIII and XhoI were purchased from New England Biolabs. T4 DNA Ligase and T4 DNALigase buffer were purchased from Takara Bio Company. Lipofectamine 3000 transfection reagent was purchased from Thermo Fisher Scientific. The commercial antibodies used in the present invention i...

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Abstract

The invention discloses a construction method of a porcine RIPK3 gene deletion cell strain capable of promoting proliferation of pseudorabies virus as well as a product and application of the porcine RIPK3 gene deletion cell strain, and relates to the technical field of biology. A construction method of a vector for constructing the cell strain comprises the following steps: annealing gRNA1-F and gRNA1-R to form a double strand 1, and connecting the double strand 1 with a CRISPR/Cas9 vector subjected to BbsI enzyme digestion to obtain pX459-gRNA1; the gRNA2-F and the gRNA2-R are annealed to form a double strand 2, and the double strand 2 is connected with an auxiliary vector subjected to BbsI enzyme digestion to obtain EZ-gRNA2; and respectively carrying out double enzyme digestion on the obtained pX459-gRNA1 and EZ-gRNA2 by using HindIII and XhoI, recovering linear enzyme digestion products, and then connecting to obtain the vector. The porcine RIPK3 gene-deleted cell strain constructed by using the vector disclosed by the invention can promote the proliferation of pseudorabies virus.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for constructing a porcine-derived RIPK3 gene-deleted cell line capable of promoting the proliferation of pseudorabies virus, its product and its application. Background technique [0002] Porcine pseudorabies virus (Pseudorabies Virus, PRV) belongs to the family Herpesviridae, subfamily alpha-herpesvirus, with a double-stranded linear DNA of 143kb, encoding more than 70 proteins. The natural host of PRV is pigs, but it can infect most mammals, including pigs, cattle, horses, rodents, and dogs, and is extremely harmful to the breeding industry. In recent years, human infection with PRV has been found many times. PRV establishes lifelong latent infection in the trigeminal ganglia of the porcine peripheral nervous system. In some cases, PRV can be reactivated, resulting in recurrent PRV epidemics in pig farms that are difficult to control and eradicate. At present, the prev...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/66C12N15/54C12N5/10C12N15/113C12N7/00
CPCC12N15/85C12N9/1205C12N15/113C12N7/00C12N2310/20C12N2710/16752
Inventor 勾红潮李春玲谢思豪卞志标翟少伦蔡汝健楚品品蒋智勇张昆丽李艳宋帅杨冬霞
Owner INST OF ANIMAL HEALTH GUANGDONG ACADEMY OF AGRI SCI
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