利用CRISPR/Cas9系统构建SBNO2基因敲除细胞系及其功能研究  

Construction of SBNO2 knockout cell lines using CRISPR/Cas9 system and its function evaluation

作　　者：王妍鳕 任亭亭 孙跃峰 刘磊[1] WANG Yanxue;REN Tingting;SUN Yuefeng;LIU Lei(College of Veterinary Medicine,Gansu Agricultural University,Lanzhou 730070,China;State Key Laboratory of Animal Etiological Biology,Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730046,China)

机构地区：[1]甘肃农业大学动物医学院,甘肃兰州730070 [2]中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室,甘肃兰州730046

出　　处：《甘肃农业大学学报》2021年第1期22-28,共7页Journal of Gansu Agricultural University

基　　金：中国农业科学院兰州兽医研究所基本科研业务费项目(1610312016010).

摘　　要：【目的】草莓缺口同源物2(Strawberry Notch Homolog 2,SBNO2)是参与炎症反应的重要基因之一,利用CRISPR/Cas9基因编辑技术靶向敲除BHK-21细胞中SBNO2基因,初步探索SBNO2基因敲除后对口蹄疫病毒(FMDV)复制的影响.【方法】首先应用Western Blot方法检测FMDV感染的BHK-21细胞中SBNO2的表达情况.然后根据SBNO2基因设计三组sgRNA,分别插入pSpCas9-puro-2A载体,构建CRISPR/Cas9-SBNO2-sgRNA重组质粒,将重组质粒转染BHK-21细胞.通过扩增基因组SBNO2序列进行测序和Western Blot验证经嘌呤霉素筛选的细胞系中SBNO2基因的敲除情况.最终利用BHK-21-SBNO2基因敲除细胞系检测其对FMDV复制的影响.【结果】sgRNA成功插入pSpCas9-puro-2A载体,Western Blot验证敲除细胞系中SBNO2的蛋白表达下调;扩增SBNO2基因序列的测序结果表明在该基因编辑位点产生移码突变.敲除细胞系感染FMDV后,qRT-PCR结果显示FMDV结构蛋白VP1表达水平显著上调.【结论】利用CRISPR/Cas9技术成功获得了BHK-21-SBNO2敲除细胞系,并发现SBNO2基因具有抑制FMDV复制的作用,为后续研究SBNO2基因作用于FMDV复制的分子机制奠定了基础.【Objective】Strawberry Notch Homolog 2(SBNO2)is one of the important genes involved in inflammatory response.To explore the role of SBNO2 gene on the replication of foot-and-mouth disease virus(FMDV),CRISPR/Cas9 gene editing technology was used to knockout SBNO2 gene in BHK-21 cells.【Method】Firstly,Western Blot was used to detect the expression level of SBNO2 in BHK-21 cells infected with FMDV.Then three sets of sgRNAs were designed according to SBNO2 gene,and inserted into pSpCas9-puro-2A vector respectively.CRISPR/cas9-SBNO2-sgRNA recombinant plasmids were constructed,and transfected into BHK-21 cells.To detect whether SBNO2 gene was knockout in the cell lines selected by puromycin,the SBNO2 sequence in the genome was amplified and sequenced,as well as Western Blot was used.Finally,BHK-21-SBNO2 gene knockout cell lines were challenged with FMDV to investigate the effect of SBNO2 gene deletion on FMDV replication.【Result】CRISPR/Cas9-SBNO2-sgRNA plasmids were constructed successfully.The stable BHK-21 cell lines of SBNO2 knockout were selected successfully.The SBNO2 protein expression in cell lines was down-regulated by Western Blot validation.The sequencing results of amplified SBNO2 gene showed that a code shift mutation occurred at the gene editing site.After infected with FMDV,compared with control cell line,SBNO2 knockout cell lines showed up-regulated expression of FMDV structural protein VP1 by qRT-PCR.【Conclusion】This study successfully obtained the stable BHK-21 cell lines of SBNO2 knockout by CRISPR/Cas9 technology.The SBNO2 gene could inhibit FMDV replication,which laid a foundation for further mechanism investigation of SBNO2 on FMDV replication.

关 键 词：CRISPR/Cas9技术 口蹄疫病毒 SBNO2基因 

分 类 号：S852.4[农业科学—基础兽医学]