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.
出处 《甘肃农业大学学报》 CAS CSCD 北大核心 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).
