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Crispr/cas9 and cre/lox system editing pseudorabies virus genome preparation method and application

A pseudorabies virus, virus vaccine technology, applied in biochemical equipment and methods, antiviral agents, viruses/phages, etc., can solve the problems of lack, wide application of virus vaccines, no multi-gene knockout, etc., to reduce disease losses , shorten the operation steps and time, and improve the efficiency

Active Publication Date: 2019-08-06
武汉都为康生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the lack of powerful gene knock-in tools, the Cre / lox system as an efficient gene knock-out tool has not been widely used in the preparation of virus vaccines
There is no report on the use of the Cre / lox system to achieve multiple gene knockouts in one step to prepare multiple gene deletion vaccines

Method used

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  • Crispr/cas9 and cre/lox system editing pseudorabies virus genome preparation method and application
  • Crispr/cas9 and cre/lox system editing pseudorabies virus genome preparation method and application
  • Crispr/cas9 and cre/lox system editing pseudorabies virus genome preparation method and application

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Embodiment 1

[0078] The preparation of the pseudorabies double gene edited virus vaccine strain of embodiment 1 deletion gE gene and TK gene

[0079] 1. Isolation and purification of wild-type pseudorabies virus

[0080] 2. Design of pseudorabies virus gE gene and TK gene sgRNA

[0081] Select 5'-GN(20)GG, 5'-GN(18)GG, 5'-GN(17)GG, 5'-N(21)GG, 5'-N(20)GG on gE gene and TK gene )GG or 5'-N(19)GG sequence site, determined by BLAST tool comparison, the selected sgRNA target sequence is the only site in the viral genome, try to avoid the possibility of off-target, gE-sgRNA and TK- The sequences of sgRNA are SEQ ID NO.1 and SEQ ID NO.2 respectively;

[0082]3. Construction of pseudorabies virus gE gene and TK gene sgRNA expression vector

[0083] Based on the sequences of gE-sgRNA and TK-sgRNA respectively, add CACC to the 5' end of the forward oligonucleotide Forward oligo, add AAA to the 5' end of the reverse oligonucleotide Reverse oligo, and design a single strand Oligonucleotides:

[...

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Abstract

The invention discloses a method for quickly preparing a vaccine by editing pseudorabies virus genomes based on a CRISPR / Cas9 gene editing system and a Cre / lox recombination system and an application of the method. According to the method, the CRISPR / Cas9 gene editing system is used for synchronously and efficiently recombining a GFP gene and a mCherry gene to a pseudorabies virus gE gene site and a TK gene site respectively to obtain conditional deletion strains of a gE gene and a TK gene; after purification, the Cre / lox system is used for cutting off extraneous GFP and mCherry genes in the pseudorabies virus recombinant virus genome so as to perform purification quickly to obtain a live pseudorabies virus vaccine lack of gE / TK genes; multiple genes are operated at the same time, so that multiple rounds of flows for knocking out multiple genes in the conventional method are reduced to one round; and meanwhile, the efficient edition of virus genes by using the CRISPR / Cas9 and Cre / lox systems simplifies about thirty generations of plaque purification processes into 3-4 generations, so that the preparation efficiency of the virus vaccine is greatly improved, and the method provides a strong guarantee for effectively preventing and controlling the larger-range popularization of variant pseudorabies viruses and reducing heavy economic losses.

Description

technical field [0001] The present invention relates to the field of preventive veterinary medicine, and specifically refers to a method for rapidly preparing vaccines based on CRISPR / Cas9 gene editing system and Cre / lox recombination system editing pseudorabies virus genome and its application. Background technique [0002] Pseudorabies virus is a member of α-herpes virus, and its genome consists of double strands of DNA. Pseudorabies virus can induce a variety of acute infectious diseases in domestic and wild animals, causing fever, itching, encephalomyelitis and other symptoms [1] . The natural host of the virus is pigs, and it can infect pigs of all ages. It is one of the most important pathogens that endanger the pig breeding industry in my country and the world. Vaccination is the most effective means of prevention and control of pseudorabies. Currently, there are a variety of commercial pseudorabies virus vaccines on the market - attenuated vaccines, inactivated vac...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/01C12N15/85A61K39/245A61P31/22
Inventor 曹罡梁勋何启盖傅振芳孙乐强余腾朱琦曹云兹
Owner 武汉都为康生物科技有限公司
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