RALY基因敲除PK-15细胞系的构建及对口蹄疫病毒复制的影响  

Construction of RALY gene knockout PK-15 cell line and its effect on replication of foot-and-mouth disease virus

作　　者：张林 吴金恩[2] 任玫 王学飞 李硕 王嘉宁 魏凡华 郭慧琛[2,3] ZHANG Lin;WU Jin-en;REN Mei;WANG Xue-fei;LI Shuo;WANG Jia-ning;WEI Fan-hua;GUO Hui-chen(College of Agriculture,Ningxia University,Yinchuan 750021,China;State Key Laboratory for Animal Disease Control and Prevention,College of Veterinary Medicine,Lanzhou University,Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730046,China;College of Veterinary Medicine,Gansu Agricultural University,Lanzhou 730070,China)

机构地区：[1]宁夏大学农学院,宁夏银川750021 [2]中国农业科学院,兰州兽医研究所,动物疫病防控全国重点实验室,兰州大学动物医学院,甘肃兰州730046 [3]甘肃农业大学动物医学院,甘肃兰州730070

出　　处：《中国兽医科学》2023年第9期1115-1121,共7页Chinese Veterinary Science

基　　金：国家重点研发计划项目(2021YFD1800300);甘肃省科技重大专项(21ZD3NA001);国家自然科学基金项目(32072859,32072847,32002272);科技人才与平台计划项目(202205AF150007)。

摘　　要：利用CRISPR/Cas9技术建立敲除RNA结合蛋白(RALY)基因的PK-15细胞系PK-15-RALY^(-/-),并初步探究RALY对口蹄疫病毒(FMDV)复制的影响。根据GenBank中猪的RALY基因的外显子,设计合成sgRNA并克隆至PX459载体;将质粒转染PK-15细胞,并采用有限稀释法筛选单克隆细胞株,经Western-blot及测序检测RALY基因的敲除效果。通过RT-qPCR检测FMDV感染PK-15-RALY^(-/-)和PK-15细胞后FMDV m RNA的转录水平,Western-blot检测FMDV-VP0、FMDV-VP3和FMDV-VP1的表达水平,最后检测子代病毒感染力。测序结果证实RALY基因发生了移码突变,且Western-blot无法检测到PK-15-RALY^(-/-)细胞中RALY蛋白的表达。FMDV感染两种细胞后,RT-PCR结果显示,FMDV在PK-15细胞中的m RNA水平显著低于PK-15-RALY^(-/-)细胞,Western-blot结果显示,VP0、VP3和VP1蛋白在PK-15-RALY^(-/-)细胞中的表达显著高于PK-15细胞,病毒感染力测定结果显示,PK-15-RALY^(-/-)细胞中子代病毒滴度显著高于PK-15细胞。上述结果表明,本研究成功构建了RALY基因敲除的PK-15细胞系,首次表明RALY可以抑制FMDV复制,为进一步开展RALY在细胞内调控病毒复制机制研究奠定了基础。RALY^(-/-)PK-15 cell line was established by knockout of RNA binding protein(RALY)gene using CRISPR/Cas9 technique,and was used to investigate the effect of RALY on the replication of foot-and-mouth disease virus(FMDV).Based on the exons of the RALYgene from pigs in GenBank,sgRNA was designed,synthesized,and cloned into the PX459 vector.The plasmids were transfected into PK-15 cells and monoclonal cell lines were screened using limited dilution method.The knockout effect of the RALY gene was detected by Western-blot and subsequently sequencing.RT-qPCR was used to detect the transcription level of FMDV mRNA in FMDV-infected PK-15-RALY^(-/-)and virus-infected control cells.Western-blot was used to detect the expression levels of FMDVVP0,FMDV-VP3,and FMDV-VP1 proteins.Finally,the infectivity of progeny virus was tested.The sequencing results confirmed the frameshift mutation of RALY gene and the expression of RALY protein in PK-15-RALY^(-/-)cells byWestern-blot.After FMDV infection with two kinds of cells,RT-PCR results showed that the mRNA level of FMDV in PK-15 cells was significantly lower than that in virus-infected PK-15-RALY^(-/-)cells,Western-blot results showed that the expression ofVP0,VP3 and VP1 proteins in virus-infected PK-15-RALY^(-/-)cells was significantly higher than that in virus-infected PK-15 cells,and the viral infectivity test results showed that progeny virus titer in virus-infected PK-15-RALY^(-/-)cells was significantly higher than that in virus-infected PK-15 cells.In this study,RALY^(-/-)PK-15 cell line was successfully constructed,which shows for the first time that RALY can inhibit the replication of FMDV,laying a foundation for further research on the mechanism of RALY in regulating virus replication.

关 键 词：CRISPR/Cas9 RALY基因 基因敲除 口蹄疫病毒 

分 类 号：S852.659.6[农业科学—基础兽医学]