利用CRISPR/Cas9基因编辑技术构建伪狂犬病病毒UL21基因缺失重组病毒  

Construction of recombinant pseudorabies virus with UL21 gene deletion using the CRISPR/Cas9 gene-editing technology

作　　者：马梓承 刘星[1] 白娟[1] 高雁怩 姜平[1] MA Zicheng;LIU Xing;BAI Juan;GAO Yanni;JIANG Ping(Nanjing Agriculture University/Key Laboratory of Animal Bacteriology,Ministry of Agriculture and Rural Affairs,Nanjing 210095,China)

机构地区：[1]南京农业大学/农业农村部动物细菌学重点实验室,江苏南京210095

出　　处：《畜牧与兽医》2023年第6期101-107,共7页Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学基金项目(32272985);国家现代农业产业技术体系专项(CARS-35)。

摘　　要：旨在构建伪狂犬病病毒(PRV)UL21基因缺失重组病毒。采用规律间隔成簇短回文重复序列/相关蛋白系统9(CRISPR/Cas9)基因编辑技术,设计针对UL21的单导向RNA(sgRNA),并构建UL21基因左右两侧同源臂质粒(pUC19 UL21-Left-Right-GFP),将其与PRV基因组共转染HEK-293T细胞,通过蚀斑纯化获得PRV-ΔUL21病毒,并通过PCR和Western blot检测方法鉴定该毒株成功缺失了UL21基因。结果表明,CRISPR/Cas9技术可用于构建PRV基因缺失重组病毒,为PRV致病机制研究提供重要依据。In order to construct a UL21 gene deletion recombinant virus of PRV,the CRISPR/Cas9 genome editing technology was used here to design an sgRNA targeting UL21,and a Left&Right homologous arms plasmid of the UL21 gene(pUC19 UL21-left-right-GFP)were constructed.Then,the above-mentioned plasmid and the PRV genome DNA were co-transfected into HEK-293T cells.The recombinant PRV with UL21 gene deletion(PRV-ΔUL21)was obtained by plaque purification,which was also confirmed by PCR and Western blot.The results indicated that the CRISPR/Cas9 technology could be used to construct recombinant pseudorabies viruses with deletions of the viral gene.This study laid an important foundation for future research on the pathogenic mechanism of PRV.

关 键 词：伪狂犬病病毒 UL21 CRISPR/Cas9 基因缺失 

分 类 号：S852.65[农业科学—基础兽医学]