靶向非洲猪瘟病毒EP152R基因sgRNA细胞系的构建及其对ASFV复制影响的研究  

Construction of sg RNA cell lines targeting the EP152R gene of African swine fever virus and its effect on ASFV replication

作　　者：皇甫皓月 雷薪霖 席飞 黄炼榆 王可欣 沈冬冬 张纪文 张振江 李芳 步志高[1] 殷昊 赵东明 HUANG FU Hao-yue;LEI Xin-lin;XI Fei;HUANG Lian-yu;WANG Ke-xin;SHEN Dong-dong;ZHANG Ji-wen;ZHANG Zhen-jiang;LI Fang;BU Zhi-gao;YIN Hao;ZHAO Dong-ming(State Key Laboratory for Animal Disease Control and Prevention,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China;Institute of Medical Sciences,Wuhan University,Wuhan 430071,China)

机构地区：[1]中国农业科学院哈尔滨兽医研究所动物疫病防控全国重点实验室,黑龙江哈尔滨150069 [2]武汉大学医学研究院,湖北武汉430071

出　　处：《中国预防兽医学报》2023年第5期452-458,共7页Chinese Journal of Preventive Veterinary Medicine

基　　金：兽医生物技术国家重点实验室自主课题(SKLVBP202101)。

摘　　要：有报道证实,缺失重要功能基因EP152R的非洲猪瘟病毒(ASFV)不能有效复制。为了筛选靶向ASFV EP152R基因的小向导RNA(sgRNA),分析EP152R基因对ASFV复制的影响,本研究利用簇状规则间隔的短回文重复序列及其相关蛋白9(CRISPR-Cas9)核酸酶系统(CCTop)并利用猪的基因组作为脱靶对象设计并筛选靶向ASFV EP152R基因(72387nt~72845nt)的sg RNA,并采用非随机整合报告系统(TIDE)计算各对sg RNA的编辑效率确定最佳sg RNA。结果显示,筛选了5对靶向敲除ASFV EP152R基因的sg RNA,其中3对sg RNA对EP152R基因的编辑效率达30%~42%,另外两对sg RNA的编辑效率低于10%。将筛选的3对sg RNA退火后分别克隆至p-LentiCRISPRv2载体中,构建3个重组慢病毒质粒p-LentiCRISPRv2-gREP152R-1/2/3,并采用菌液PCR及测序鉴定正确后分别与2个辅助质粒共转染HEK293T细胞,72 h后获得各慢病毒并分别感染野猪肺细胞(WSL细胞),通过嘌呤霉素筛选表达各sg RNA的细胞WSL-g REP152R,并采用western blot鉴定。结果显示,构建的各细胞在40 ku处均出现特异性条带,而WSL细胞无该条带,表明正确构建表达3种EP152R sg RNA的细胞系WSL-g REP152R-1/2/3。将携带m Cherry报告基因的重组ASFV(mCherry-ASFV)以MOI 1感染各WSL-g REP152R细胞系,分别于不同时间观察荧光;收集各细胞上清液,采用荧光定量PCR(qPCR)检测ASFV P72基因的Ct值,并测定上清液中的病毒效价(感染后72 h)。观察结果显示,随感染时间的延长WSL-g REP152R-2/3中的红色荧光均明显减少;WSL-g REP152R-1及空白对照WSL细胞中的红色荧光则逐渐增多,但前者的红色荧光均少于后者。q PCR和TCID50测定结果显示,与空白对照WSL细胞相比,3种WSL-g REP152R细胞上清液中病毒扩增的Ct值均显著或极显著升高(P<0.05、P<0.01),而病毒效价均显著或极显著降低(P<0.05、P<0.01),且以WSL-g REP152R-2中的病毒效价最低。将m Cherry-ASFV感染各WSLg REP152R细胞并连续传3代,采用q PCR检测F2Reports have confirmed that African swine fever virus(ASFV) lacking the important functional gene EP152R cannot replicate efficiently.In order to screen small guide RNAs(sgRNAs) targeting the ASFV EP152R gene and analyze the effect of the EP152R gene on ASFV replication,we designed and screened sg RNAs targeting the ASFV EP152R gene(72387nt-72845nt) by using a clustered regularly interspaced short palindromic repeats(CRISPR)-Cas9 nuclease system(CCTop) and utilizing the porcine genome as an off-target object.The best sg RNA targeting gene(72387nt-72845nt) was identified by calculating the editing efficiency of each pair of sg RNAs using a non-randomized integrated reporter system(TIDE).The results showed that five pairs of sg RNAs targeting the knockdown of the ASFV EP152R gene were screened,and three pairs of the sg RNAs had editing efficiencies ranging from 30% to 42%.In contrast,the editing efficiency of the other two pairs of sg RNAs was lower than 10%.Therefore,the three pairs of sg RNAs were cloned into p-LentiCRISPRv2 vector after annealing,and recombinant lentiviral plasmids p-LentiCRISPRv2-gREP152R-1/2/3 were constructed.The correct ones,confirmed by bacteriophage PCR and sequencing,were co-transfected with the two helper plasmids into HEK293T cells.Each lentivirus obtained 72 hours after transfection was used for infection of wild boar lung WSL cells respectively.And the cells WSLg REP152R expressing each sg RNA were screened by puromycin and identified by western blot.The constructed three cell lines WSL-g REP152R-1/2/3 showed a specific band at 40ku,while no protein expression at 40ku was observed in WSL cells,indicating that cell lines expressing the three EP152R sg RNAs were correctly constructed.Each WSL-g REP152R cell line was infected with recombinant ASFV(mCherry-ASFV) carrying the m Cherry reporter at an MOI 1,and the fluorescence was observed at different times.The supernatant of each cell line was collected,and the Ct value of the ASFV P72 gene was detected by fluorescence quantitative PCR(qP

关 键 词：非洲猪瘟病毒 簇状规则间隔的短回文重复序列及其相关蛋白9 EP152R g RNA 

分 类 号：S852.65[农业科学—基础兽医学]