基于SYNJ2BP敲除的HEK293T细胞系研究SYNJ2BP对EIAV复制的影响  

Construction of SYNJ2BP-knockout HEK293T cell line and its effect on there plication of equine infectious anemia virus

作　　者：王欣慧 段盈伊 陈克伟 王雪峰[1] 那雷[1] 杜承[1] 王晓钧[1] WANG Xin-hui;DUAN Ying-yi;CHEN Ke-wei;WANG Xue-feng;NA Lei;DU Cheng;WANG Xiao-jun(State Key Laboratory of Veterinary Biotechnology,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Heilongjiang Province,Harbin 150069,China)

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室,黑龙江哈尔滨150069

出　　处：《中国预防兽医学报》2021年第12期1258-1262,1281,共6页Chinese Journal of Preventive Veterinary Medicine

基　　金：黑龙江省自然科学基金面上项目(C2018071);国家自然科学基金面上项目(31772720)。

摘　　要：为分析突触小泡磷酸酶2结合蛋白(SYNJ2BP)对马传染性贫血病毒(EIAV)复制的影响,本研究设计了2条靶向SYNJ2BP基因外显子的特异性sgRNA,将sgRNA插入plenti-CRISPRv2GFP载体获得重组质粒pSYNsgRNA1和pSYN-sgRNA2,将这2个重组质粒转染HEK293T细胞,通过流式细胞仪分选获得单克隆细胞株,测序结果显示SYNJ2BP基因产生插入突变,经western blot确认已敲除SYNJ2BP基因并稳定遗传,表明获得了SYNJ2BP敲除的HEK293T细胞系。将EIAV感染性克隆质粒CMV3-8分别转染SYNJ2BP敲除的HEK293T细胞系与野生型细胞,经western blot证实与野生型细胞相比敲除SYNJ2BP抑制了EIAV囊膜蛋白的表达,灰度值显示囊膜蛋白表达量降低50%。综上所述,本研究利用CRISPR/Cas9基因编辑技术构建了SYNJ2BP敲除的HEK293T细胞系,并发现SYNJ2BP基因敲除后抑制了EIAV囊膜蛋白的表达,本研究为进一步分析SYNJ2BP调节EIAV复制的分子机制提供了参考依据。To identify the role of synaptojanin 2 binding protein(SYNJ2 BP)on equine infectious anemia virus(EIAV)replication and establish SYNJ2 BP genek nockout HEK239 T cell lines,two specific single guide RNAs(sgRNAs)were designed to target exons of SYNJ2 BP gene.SgRNA was inserted into plenti-CRISPRv2 GFP vector to obtain the recombinant plasmids pSYNsgRNA1 and pSYN-sgRNA2,respectively.HEK293 T cells were transfected with two recombinant plasmids.The transduced HEK293 T cells were sorted by High-speed Cell Sorter to obtain a single-cell-derived colony.The sequencing analysis showed that insertion mutation occurs in the SYNJ2 BP gene.Western blot results demonstrated that the SYNJ2 BP gene was successfully knocked out and the heredity was stable.Subsequently,SYNJ2 BP-knockout HEK293 T cell lines and wild-type cells were transfected with CMV3-8(an infectious clone of EIAV)and the replication level of EIAV was evaluated by western blot.The results showed that SYNJ2 BP gene knockout inhibited the formation of envelope protein of EIAV,and its relative intensity reduced50%.In summary,this study established SYNJ2 BP-knockout EK293 T cell lines using CRISPR/Cas9 system and found that SYNJ2 BP-knockout HEK293 T inhibited the expression of envelop protein of EIAV,which provides foundations for further study of the molecular mechanism of SYNJ2 BP regulating EIAV replication.

关 键 词：CRISPR/Cas9 马传染性贫血病毒 SYNJ2BP HEK293T 

分 类 号：S852.65[农业科学—基础兽医学]