应用CRISPR/Cas9基因编辑系统敲除小窝蛋白1基因的神经细胞构建与功能鉴定  

Constructing and functional analysis of knocking out CAV-1 gene by CRISPR/Cas9 system in neuron cells

作　　者：权力 江爱民[1,3] 卢彦宗 朱耐伟 朱勇喆 QUAN Li;JIANG Ai-min;LU Yan-zong;ZHU Nai-wei;ZHU Yong-zhe(Department of Microbiology,Second Military Medical University,Shanghai 200433,China;Companyl,Cadet＇s Brigade,Second Military Medical University;Company 9,Cadet＇s Brigade,Second Military Medical University)

机构地区：[1]第二军医大学微生物学教研室,上海200433 [2]第二军医大学学员旅学员1队,上海200433 [3]第二军医大学学员旅学员9队,上海200433

出　　处：《中国媒介生物学及控制杂志》2018年第5期453-457,共5页Chinese Journal of Vector Biology and Control

基　　金：第二军医大学创新能力培养计划(FH2016123;FH2016124)~~

摘　　要：目的利用CRISPR/Cas9基因编辑系统建立小窝蛋白1(CAV-1)基因敲除的神经母细胞瘤细胞株(SH-SY5Y),并验证其对流行性乙型脑炎病毒(JEV)的易感性。方法根据CRISPR/Cas9靶点设计原则,基于人源CAV-1基因的外显子区(EXON)序列设计小向导RNA(sgRNA),将sgRNA连接到载体PX459质粒上。将重组质粒转染至SHSY5Y细胞中,使用嘌呤霉素筛选稳定敲除CAV-1蛋白的SH-SY5Y细胞,并用免疫印迹法验证CAV-1蛋白的敲除效果。免疫荧光法比较CAV-1基因敲除的SH-SY5Y株与野生型细胞株对JEV感染性的差异。结果经酶切和测序鉴定,重组真核表达质粒构建正确,转染并筛选的单克隆细胞中没有CAV-1蛋白的表达,成功构建CAV-1基因敲除的SH-SY5Y细胞株。与野生型SH-SY5Y细胞株相比,CAV-1基因敲除的细胞株对JEV的感染性下降约90%(P<0.001)。结论利用CRISPR/Cas9系统成功构建CAV-1基因敲除的SH-SY5Y细胞株,该细胞株可用于进一步研究CAV-1蛋白在JEV感染中的作用机制。Objective To establish a stable CAV-1 gene knockout cell line by CRISPR/Cas9 system in neuroblastoma cellline（SH-SY5 Y）, and to investigate its function on Japanese encephalitis virus（JEV） infection. Methods The sgRNAsequences targeting EXON of human CAV-1 gene were designed according to the principles of CRISPR/Cas9, and thenwere cloned into PX459 plasmid.The SH-SY5 Y cells transfected with the recombinant plasmids were selected bypuromycin to gain the CAV-1 knockout cells. The cells with CAV-1 knockout effect were verified by Western blotting. Thedifferences of JEV infection on CAV-1 knockout cells and wild type cells were detected by immunofluorescence. Results The recombinant plasmids were verified by enzyme cutting and gene sequencing and were successfully constructed. Theprotein of CAV-1 was undetected in SH-SY5 Y cells after transfection and screening of monoclonal cell by puromycin andthe CAV-1 knockout SH-SY5 Y cells were successfully constructed. Compared to the wild type cells, the infectivity of JEVon the CAV-1 knockout cells decreased by 90%（P〈0.001）. Conclusion The CAV-1 gene was knocked out successfullyby CRISPR/Cas9 system in SH-SY5 Y cells. This cell line can be used to further study the mechanism of JEV infection.

关 键 词：基因编辑 小窝蛋白 神经细胞 流行性乙型脑炎病毒 

分 类 号：R373.3[医药卫生—病原生物学] Q78[医药卫生—基础医学]