利用CRISPR/Cas9系统快速构建携带EGFP的PRV-ΔTK重组变异株
Rapid construction of rPRV-ΔTK/EGFP variant strain using CRISPR/Cas9 sys-tem
叶再娇 1曾川 2顾俊 2王培霞 2沈金燕 2宋德平 2黄冬艳 2邬向东 2何后军 2唐玉新 2叶昱2
作者信息
- 1. 江西农业大学 动物科学技术学院,江西 南昌 330045;江西省动物疫病防控制剂工程研究中心,江西 南昌 330045;江西农业大学 工学院,江西 南昌 330045
- 2. 江西农业大学 动物科学技术学院,江西 南昌 330045;江西省动物疫病防控制剂工程研究中心,江西 南昌 330045
- 折叠
摘要
伪狂犬病病毒(pseudorabies virus,PRV)能够引起猪只出现以呼吸困难、繁殖障碍和神经系统疾病等为特征的伪狂犬病,在世界范围内传播广泛.2011年以来,新现的PRV变异株导致传统疫苗株的免疫保护效果不佳,且原有的疫苗株制备方法耗时耗力,因此现阶段急需开发一种高效疫苗株筛选的方法.采用CRISPR/Cas9基因编辑技术,设计2条靶向PRV毒力基因TK的单链引导RNA(single guide RNA,sgRNA),敲除PRV变异株CH/JX/2016编码的TK基因,再利用同源修复质粒在TK基因座位置插入增强绿色荧光蛋白(enhanced green fluorescent protein,EGFP),经多轮蚀斑纯化获得rPRV-ΔTK/EGFP毒株.结果表明本研究构建的sgRNA切割效率高,只经过3轮纯化就可完成rPRV-ΔTK/EGFP毒株的制备,且EGFP基因正常表达.CRISPR/Cas9系统能够简便快速高效地编辑PRV基因,在疫苗候选株创制中潜力巨大,而且拯救的rPRV-ΔTK/EGFP毒株不仅可以作为研究PRV变异株感染进程的示踪毒株,还能用于后续抗病毒药物的筛选.
Abstract
Pseudorabies virus(PRV)is the etiological agent of pseudorabies in pigs,which is char-acterized by dyspnea,reproductive disorders,and neurological diseases,and it spreads widely a-round the world.Since 2011,the newly emerged PRV variants have resulted in poor immunity pro-tection of traditional vaccine strains,and the original method of vaccine strain preparation is time-consuming and labor-intensive.Therefore,it is urgently needed to develop an efficient screening method of the vaccine strain at present.Using CRISPR/Cas9 gene editing technology in this study,two single guide RNAs(sgRNA)were designed targeting the virulence gene TK of PRV variant strain CH/JX/2016,and then the enhanced green fluorescent protein the reporter(EGFP)gene was inserted at the TK locus by a homologous repair plasmid.After multiple rounds of plaque puri-fication,the rPRV-ΔTK/EGFP strain was obtained.The results showed the cleavage efficiency of the two sgRNAs was extremely high.The preparation of rPRV-ΔTK/EGFP strain was succeed af-ter only three rounds of purification,and the EGFP expressed normally.The CRISPR/Cas9 system can edit the PRV gene simply,rapidly,and efficiently,and exhibits great potential in the construction of vaccine candidate strains.Meanwhile,the rescued rPRV-ΔTK/EGFP strain not only could be used as a tracer strain in PRV variant infection progresses,but also for subsequent antivi-ral drug screening.
关键词
伪狂犬病病毒/CRISPR/Cas9/增强绿色荧光蛋白/重组/蚀斑纯化Key words
PRV/CRISPR/Cas9/EGFP/recombination/plaque purification引用本文复制引用
基金项目
国家自然科学基金(32002289)
江西省科技计划(20221ZDH04055)
江西省科技计划(20203BBF63020)
出版年
2024
NETL
NSTL
万方数据