针对非洲猪瘟病毒p30基因CRISPR/Cas9基因编辑系统的构建和初步鉴定  

Construction and Identification of CRISPR/Cas9 Gene Editing System Targeting African Swine Fever Virus p30 Gene

作　　者：于海男 崔帅 王洋 刘雪婷[1] 高新桃 房立春 郭晓宇[1] 朱鸿飞[1] YU Hainan;CUI Shuai;WANG Yang;LIU Xueting;GAO Xintao;FANG Lichun;GUO Xiaoyu;ZHU Hongfei(Institute of Animal Science,CAAS,Beijing 100193,China;Biotechnology Research Institute,CAAS,Beijing 100081,China;Poultry Institute,Shandong Academy of Agricultural Sciences,Jinan 250031,China)

机构地区：[1]中国农业科学院北京畜牧兽医研究所,北京100193 [2]中国农业科学院生物技术研究所,北京100181 [3]山东省农业科学院家禽研究所,济南250031

出　　处：《中国动物传染病学报》2023年第2期59-66,共8页Chinese Journal of Animal Infectious Diseases

基　　金：国家重点研发计划课题(2018YFC0840404);山东省重大科技创新工程(2020CXGC010801)。

摘　　要：构建针对非洲猪瘟病毒(ASFV)p30(CP204L)基因的CRISPR/Cas9基因编辑系统,探究该系统对p30蛋白真核表达的影响。以含p30基因序列的质粒p30-IRES为模板,定向扩增目的基因片段并克隆至pmCherry-N1载体,构建含有p30、红色荧光报告基因的融合表达质粒;通过网站http://crispr.mit.edu/设计靶向CP204L的单导向RNA(sgRNA),并克隆至Cas9表达质粒eSpCas9-2A-GFP(PX458)中。将融合表达质粒与Cas9表达质粒共转染HEK 293T细胞,同时设置对照组,转染48 h后通过Western blot和荧光定量PCR(qPCR)方法分别检测转染细胞中p30蛋白和mRNA表达水平。由于CRISPR/Cas9基因编辑系统靶向切割p30基因,Western blot和qPCR结果显示转染靶向p30基因的Cas9质粒后,p30蛋白的真核表达受到抑制,具有显著性差异(P<0.05)。结果表明,CRISPR/Cas9基因编辑系统可以高效切割非洲猪瘟p30基因,抑制该蛋白的真核表达水平,该体系为抑制病毒生长和疫情防控提供了新的研究思路。The objectives of the present study were to construct the CRISPR/Cas9 gene editing system targeting African swine fever virus(ASFV)p30(CP204L)gene and explore its effect on p30 protein eukaryotic expression.Firstly,the pmCherry-p30 recombinant red fl uorescence report plasmid with p30-IRES plasmid and pmCherry-N1 plasmid was constructed.Then the single guide RNA(sgRNA)targeting CP204L was designed through the website http://crispr.mit.edu/and cloned into the Cas9 expression plasmid eSpCas9-2A-GFP(PX458).The p30 expression plasmid and Cas9 expression plasmid were co-transfected into HEK 293T cells.The expression levels of p30 protein and mRNA in the transfected cells were detected at 48 h post-transfection using Western blot and quantitative PCR.The results showed that the Cas9 plasmid targeting the p30 gene successfully inhibited the eukaryotic expression of the p30 protein with significant differences(P<0.05).In conclusion,the CRISPR/Cas9 gene editing system reported here provided a new tool for inhibition of ASFV growth and prevention of the ASF epidemic.

关 键 词：非洲猪瘟病毒 P30基因 CRISPR/Cas9 单导向RNA 真核表达 

分 类 号：S852.651[农业科学—基础兽医学]