利用CRISPR/Cas9系统构建STING基因敲除的猪肺泡巨噬细胞系及其功能验证  

Construction of STING knockout porcine alveolar macrophages via CRISPR/Cas9 and the functional characterization of the STING^(-/-) cells

作　　者：张佳佳 王辉 夏能文 郑王龙 朱建中[1,2,3,4] ZHANG Jia-jia;WANG Hui;XIA Neng-wen;ZHENG Wang-long;ZHU Jian-zhong(College of Veterinary Medicine,Yangzhou University,Yangzhou 225009,China;Joint International Research Laboratory of Agriculture and Agri-product Safety,Yangzhou University,Yangzhou 225009,China;Comparative Medicine Research Institute,Yangzhou University,Yangzhou 225009,China;Jiangsu Co-innovation Center for Prevention angd Control of Important Animal Infections Diseases and Zoonoses,Yangzhou University,Yangzhou 225009,China)

机构地区：[1]扬州大学兽医学院,江苏扬州225009 [2]扬州大学农业与农产品安全国际联合研究实验室,江苏扬州225009 [3]扬州大学比较医学研究中心,江苏扬州225009 [4]扬州大学江苏省动物重要疫病与人兽共患病防控协同创新中心,江苏扬州225009

出　　处：《中国兽医科学》2022年第5期617-624,共8页Chinese Veterinary Science

基　　金：国家自然科学基金项目(3217190122,31872450);江苏高校优势学科建设工程资助项目(PAPD)。

摘　　要：本研究通过CRISPR/Cas9系统构建STING基因敲除的猪肺泡巨噬细胞系(PAM),并验证STING敲除细胞介导IFN-Ⅰ产生和抗单纯疱疹病毒Ⅰ型(HSV-Ⅰ)感染的功能变化。针对猪STING基因靶向设计向导RNA(sgRNA),将连接sgRNA的pX-458质粒转染PAM细胞,利用定向筛选和有限稀释法建立STING基因敲除细胞克隆。利用DNA测序和Western-blot方法分别对所获细胞克隆的STING基因和蛋白表达进行鉴定。通过RT-qPCR和Western-blot对STING敲除细胞进行功能鉴定,通过荧光显微镜观察STING敲除对HSV-Ⅰ复制的影响。扩增STING基因序列的测序结果表明,在该STING基因编辑位点产生碱基缺失移码突变,Western-blot结果显示,敲除细胞中STING蛋白表达消失。经poly(dA:dT)或2′3′-cGAMP刺激后,与正常PAM细胞相比,STING敲除的PAM细胞中刺激诱导的IFN-β和ISG56 mRNA表达下降;刺激诱导的P-TBK1、P-IRF3和ISG56蛋白表达也显著下调;同时HSV-Ⅰ复制水平显著上调。本研究通过CRISPR/Cas9技术成功构建了STING基因敲除的PAM细胞克隆,验证了STING敲除对诱导IFN-Ⅰ相关信号及抗HSV-Ⅰ感染的影响,为进一步研究STING在猪天然免疫中的作用提供了细胞工具和手段。This study used the CRISPR/Cas9 system to construct a STING knockout porcine alveolar macrophage cell line(PAM)and characterized the functional changes of STING-/-cells in mediating IFN-Ⅰproduction and resisting HSV-Ⅰinfection.Two small guide RNA(sgRNA)targeting pig STING gene exon 3 were designed and the sgRNA plasmids were transfected into PAM cells.The individual cell clones based on the GFP signal and limited dilution were obtained and the obtained cell clones were screened by PCR and DNA sequencing of STING gene,further verified for STING protein expression by Western-blot.The function changes of STING-/-cells were identified by RT-qPCR and Western-bot,and the effect of STING knockout on HSV-Ⅰreplication was observed by fluorescence microscopy.DNA sequencing results of the cloned STING PCR products showed that the base deletion frameshift mutations were generated at the STING gene editing site,and the Western-blot results showed that the expression of STING protein in STING-/-cells disappeared.After stimulation with poly(dA:dT)or 2′3′-cGAMP,compared with normal PAM cells,the expression lev els of IFN-βand ISG56 mRNA in STING-/-PAM cells decreased;the protein expressions of p-TBK1,p-IRF3 and ISG56 were also significantly downregulated.At the same time,the replication level of HSV-Ⅰwas significantly upregulated.This study used CRISPR/Cas9 technology to successfully construct a PAM-/-cell clone,and verified the effect of STING knockout on the induction of IFN-Ⅰrelated signals and resistance to HSV-1 infection.This knockout cell line will be a very useful tool to study the function of STING in the pig immunity.

关 键 词：CRISPR/Cas9 猪 天然免疫 STING 猪肺泡巨噬细胞系 基因敲除 

分 类 号：S852.42[农业科学—基础兽医学]