Method for knocking out pseudorabies virus TK gene by using dual sgRNA and application of method

A pseudorabies virus and gene technology, applied in the field of genetic engineering, can solve problems such as high off-target efficiency and instability of knockout results, and achieve the effects of simple construction process, easy screening of recombinant viruses, and increased expression

Pending Publication Date: 2021-05-11
天津市农业科学院
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] However, the CRISPR / Cas knockout method has high off-target efficiency and the knockout results are unstable

Method used

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  • Method for knocking out pseudorabies virus TK gene by using dual sgRNA and application of method
  • Method for knocking out pseudorabies virus TK gene by using dual sgRNA and application of method
  • Method for knocking out pseudorabies virus TK gene by using dual sgRNA and application of method

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Embodiment 1

[0070] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described in detail below. Apparently, the described embodiments are only some of the embodiments of the present invention, but not all of them. Based on the embodiments of the present invention, all other implementations obtained by persons of ordinary skill in the art without making creative efforts fall within the protection scope of the present invention. Embodiment 1 constructs porcine pseudorabies virus TK gene deletion strain

[0071] 1 Test material

[0072] 1.1 Tested virus strains

[0073] Porcine pseudorabies virus (PRV) R13139 is the PRV epidemic strain isolated from suspected cases of pseudorabies in pig farms in Tianjin, and the R13139-gE strain is the R13139 gE gene deletion strain prepared by the present invention.

[0074] 1.2 Main reagents

[0075] LA TaqDNA enzyme, 2XGC buffer I, dNTP, DL2000 DNA...

Embodiment 2

[0155] Embodiment 2 measures pseudorabies virus gE-TK double gene strain biological characteristic

[0156] 1. Growth curve determination

[0157] The PRV-gE-TK double gene deletion virus with a virus content of 1MOI and the PRV R13139 strain were inoculated into PK15 cells, and the supernatant was discarded after adsorption at 37°C for 1 hour, and the virus solution was harvested every 4 hours until 36 hours. The harvested virus liquid was measured for virus content respectively, and the growth curves of the two viruses were drawn. The results are shown in image 3 .

[0158] The result is as image 3 As shown, the growth curves of the PRV-gE-TK double gene deletion virus and the PRV R13139 strain virus had no significant difference, and both reached the peak of proliferation at 28 hours.

[0159] 2. Determination of Genetic Stability

[0160] The PRV-gE-TK virus was continuously passed on for 10 generations on PK15 cells, and PCR was performed on each generation to ident...

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Abstract

The invention belongs to the field of genetic engineering and particularly relates to a method for knocking out a pseudorabies virus TK gene by using dual sgRNA and application of the method. According to the method, a dual-locus knockout method is employed, two sgRNA sequences are designed on the TK gene, two loci are cut in a pointed manner, and thus, the TK gene can be efficiently knocked out. The method for knocking out the pseudorabies virus TK gene mainly comprises the procedures of sgRNA designing, Cas9 vector-sgRNA expression vector constructing, donor vector constructing, plasmid transfection, cre enzyme vector transfection and recombinant virus purification. Compared with the traditional methods, the method disclosed by the invention has the advantages of high accuracy and low target missing effect.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a method for knocking out the TK gene of pseudorabies virus by using a double sgRNA CRISPR / cas9 system and a Cre-loxp editing system and its application. Background technique [0002] The vector expression system is the key to the successful development and application of recombinant vaccines. It can not only carry foreign genes, but also express foreign genes efficiently. Currently, live viral vectors used in recombinant vaccines mainly include lentiviruses, adenoviruses, poxviruses, baculoviruses, and herpesviruses. Porcine pseudorabies virus (PRV) belongs to herpes virus. As a viral vector, it has a relatively large genome, many non-essential segments for replication, and can insert multiple foreign genes at the same time. It is an ideal multivalent recombinant vaccine vector. , which can be used to construct gene-marked vaccines to achieve multi-defense with on...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/66C12N15/65C12N15/55C12N15/38C12N7/00C12R1/93
CPCC12N15/85C12N15/65C12N9/22C07K14/005C12N7/00C12N2800/107C12N2800/30C12N2710/16722C12N2710/16752C12N2710/16721
Inventor 张莉鄢明华路超任卫科李秀丽王利丽董志民李富强田向学江珊李程
Owner 天津市农业科学院
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