中国媒介生物学及控制杂志 ›› 2018, Vol. 29 ›› Issue (5): 453-457.DOI: 10.11853/j.issn.1003.8280.2018.05.008

• 论著 • 上一篇    下一篇

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

权力1,2, 江爱民1,3, 卢彦宗1,2, 朱耐伟1, 朱勇喆1   

  1. 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
  • 收稿日期:2018-06-02 出版日期:2018-10-20 发布日期:2018-10-20
  • 通讯作者: 朱勇喆,Email:zhuyongzhe1984@sina.com
  • 作者简介:权力,男,在读博士,主要从事病毒致病机制研究,Email:13340700015@fudan.edu.cn;江爱民,男,在读本科,主要从事病毒致病机制研究,Email:18301935531@163.com
  • 基金资助:
    第二军医大学创新能力培养计划(FH2016123,FH2016124)

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

QUAN Li1,2, JIANG Ai-min1,3, LU Yan-zong1,2, ZHU Nai-wei1, ZHU Yong-zhe1   

  1. 1 Department of Microbiology, Second Military Medical University, Shanghai 200433, China;
    2 Company1, Cadet's Brigade, Second Military Medical University;
    3 Company 9, Cadet's Brigade, Second Military Medical University
  • Received:2018-06-02 Online:2018-10-20 Published:2018-10-20
  • Supported by:
    Supported by the Innovation Ability Training Program (No. 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SH-SY5Y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。

关键词: 基因编辑, 小窝蛋白, 神经细胞, 流行性乙型脑炎病毒

Abstract: Objective To establish a stable CAV-1 gene knockout cell line by CRISPR/Cas9 system in neuroblastoma cell line (SH-SY5Y), and to investigate its function on Japanese encephalitis virus (JEV) infection. Methods The sgRNA sequences targeting EXON of human CAV-1 gene were designed according to the principles of CRISPR/Cas9, and then were cloned into PX459 plasmid.The SH-SY5Y cells transfected with the recombinant plasmids were selected by puromycin to gain the CAV-1 knockout cells. The cells with CAV-1 knockout effect were verified by Western blotting. The differences 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. The protein of CAV-1 was undetected in SH-SY5Y cells after transfection and screening of monoclonal cell by puromycin and the CAV-1 knockout SH-SY5Y cells were successfully constructed. Compared to the wild type cells, the infectivity of JEV on the CAV-1 knockout cells decreased by 90% (P<0.001). Conclusion The CAV-1 gene was knocked out successfully by CRISPR/Cas9 system in SH-SY5Y cells. This cell line can be used to further study the mechanism of JEV infection.

Key words: Gene editing, Caveolin, Neuron cells, Japanese encephalitis virus

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