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Porcine MLKL gene deletion cell strain capable of promoting proliferation of pseudorabies virus and application of porcine MLKL gene deletion cell strain

A gene deletion and cell strain technology, applied to genetically modified cells, cells modified by introducing foreign genetic material, viruses, etc., can solve problems that are difficult to control and eradicate, and achieve the effect of promoting proliferation and virus proliferation

Active Publication Date: 2022-05-10
INST OF ANIMAL HEALTH GUANGDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In some cases, PRV can reactivate, leading to repeated PRV epidemics in pig farms, difficult to control and eradicate

Method used

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  • Porcine MLKL gene deletion cell strain capable of promoting proliferation of pseudorabies virus and application of porcine MLKL gene deletion cell strain
  • Porcine MLKL gene deletion cell strain capable of promoting proliferation of pseudorabies virus and application of porcine MLKL gene deletion cell strain
  • Porcine MLKL gene deletion cell strain capable of promoting proliferation of pseudorabies virus and application of porcine MLKL gene deletion cell strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Construction and screening method of a pig-derived MLKL gene-deleted cell strain that can promote the proliferation of pseudorabies virus

[0037] 1. Materials and methods

[0038] Cells, viruses and plasmids: Porcine kidney epithelial cells (PK-15) were purchased from China Center for Type Culture Collection (Wuhan University), collection number: GDC0061; PRV Bartha-K61 vaccine strain (PRVBartha-K61) was purchased from From the China Veterinary Microbial Culture Collection Management Center, preservation number: CVCC AV249; PRV GD-WH strain (PRV GD-WH) isolated and preserved from the Pig Disease Laboratory of the Institute of Animal Health, Guangdong Academy of Agricultural Sciences; Escherichia coli Trans10 competent cells purchased From Addgene Company; CRISPR / Cas9 vector plasmid pX459 pSpCas9-2Apuro-MCS and auxiliary vector plasmid EZ-GuideXH were purchased from Addgene Company.

[0039]Reagents and antibodies: Restriction endonucleases BbsⅠ, HindIII and ...

Embodiment 2

[0061] Example 2 Analysis of the promoting effect of pig-derived MLKL gene-deleted cell lines on PRV proliferation

[0062] Infect PK-15 and PK-15MLKL-KO cells with PRV GD-WH and PRV Bartha-K61 at MOI=10, respectively, and place at 37°C, 5% CO 2 The incubator adsorbed for 1h. After the adsorption, the inoculum was discarded, the cells were washed 3 times with PBS, and replaced with maintenance medium for culture. The cell supernatant virus liquid was collected according to different time points of 12h, 24h, and 36h, the virus liquid was repeatedly frozen and thawed three times, the supernatant was obtained by centrifugation, and the supernatant was diluted 10 times, respectively, and infected with PK-15 cells in a 96-well plate , observed 4 days after virus infection, and recorded the lesions of each well. The PRV GD-WH strain and PRV Bartha-K61 strain titers of each supernatant were determined according to the Reed-Muench method.

[0063] The result is as Image 6 with ...

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Abstract

The invention discloses a swine MLKL gene deletion cell strain capable of promoting proliferation of pseudorabies virus and application of the swine MLKL gene deletion cell strain, and belongs to the technical field of biology. The invention discloses sgRNA for knocking out a swine MLKL gene, which is characterized in that the sgRNA comprises sgRNA1 and sgRNA2, the primer sequence of the sgRNA1 is as shown in SEQ ID NO: 1-2, the primer sequence of the sgRNA2 is as shown in SEQ ID NO: 1-2, and the primer sequence of the sgRNA2 is as shown in SEQ ID NO: 1-2. The primer sequence of the sgRNA2 is as shown in SEQ ID NO: 3-4. The sgRNA primer and a CRISPR / Cas9 carrier are utilized to construct a knockout porcine MLKL gene, then a limited dilution method is adopted to carry out passage and screening on the obtained porcine kidney epithelial cells with the knockout porcine MLKL gene to obtain a porcine MLKL gene deletion cell strain, and after the porcine kidney epithelial cells with the knockout porcine MLKL gene deletion cell strain is inoculated with pseudorabies virus, the cell strain can continuously promote virus proliferation compared with normal porcine kidney epithelial cells.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a pig-derived MLKL gene-deleted cell strain capable of promoting the proliferation of pseudorabies virus and its application. Background technique [0002] Porcine pseudorabies virus (Pseudorabies Virus, PRV) belongs to Herpesviridae, α-herpesvirus subfamily, has 143kb double-stranded linear DNA, and encodes 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, many cases of human infection with PRV have been found. PRV can establish a lifelong latent infection in the trigeminal ganglion of the porcine peripheral nervous system. In some cases, PRV can reactivate, which in turn leads to repeated PRV epidemics in pig farms, which are difficult to control and eradicate. At present, vaccination is an important means of prevention and ...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/85C12N5/10C12N7/00
CPCC12N15/1137C12N15/85C12N9/12C12N5/0686C12N7/00C12N2310/20C12N2510/00C12N2710/16751
Inventor 勾红潮李春玲谢思豪卞志标翟少伦蔡汝健楚品品李艳蒋智勇宋帅张昆丽杨冬霞
Owner INST OF ANIMAL HEALTH GUANGDONG ACADEMY OF AGRI SCI
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