作 者：
曾为俊;许曼;王辉;鲁国涛;邵玉乐;陈洪岩;刘建华;孟庆文
关键词：
RIG-I;CRISPR/Cas9;MDCK细胞系;基因敲除;H9N2亚型流感病毒
摘 要：
利用CRISPR/Cas9技术构建RIG-I基因敲除MDCK细胞株,并验证其生物学功能.试验设计并构建了2个靶向RIG-I基因的导向RNA(sgRNA)表达载体,与pCAG-Cas9-EGFP表达载体共转染MDCK细胞,采用PAGE胶基因型检测、定点测序和Western blot筛选细胞株;通过荧光定量PCR检测H9N2亚型禽流感病毒(AIV)感染后MAVS、IRF3、IFN-β、IL-6、Mx1 mRNA表达水平和病毒基因拷贝数,测定TCID50.结果表明:获得两株稳定敲除RIG-I基因的MDCK细胞(MDCK-RIG-I-/-),Western blot检测RIG-I蛋白不表达;荧光定量PCR结果显示病毒感染后MDCK-RIG-I-/-的MAVS、IRF3、IFN-β、IL-6、Mx1 mRNA表达水平显著降低,表明RIG-I基因敲除后RIG-I-Ⅰ型干扰素信号通路受阻;病毒基因拷贝数、TCID50测定结果显示,差异最高可分别达到野生型MDCK的2.16倍、3.19倍,表明RIG-I基因敲除后流感病毒复制增加.该研究利用CRISPR/Cas9技术建立了敲除RIG-I基因流感病毒MDCK细胞株,为研究RIG-I天然免疫应答机制奠定基础;该技术方法和策略也为流感疫苗生产及产业更新换代提供候选细胞株.
作 者：
ZENG Weijun;XU Man;WANG Hui;LU Guotao;SHAO Yule;CHEN Hongyan;LIU Jianhua;MENG Qingwen;College of Animal Medicine, Xinjiang Agricultural University;Heilongjiang Provincial Key Laboratory of Laboratory Animals and Comparative Medicine,State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences;
单 位：
ZENG Weijun%XU Man%WANG Hui%LU Guotao%SHAO Yule%CHEN Hongyan%LIU Jianhua%MENG Qingwen%College of Animal Medicine, Xinjiang Agricultural University%Heilongjiang Provincial Key Laboratory of Laboratory Animals and Comparative Medicine,State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences
关键词：
RIG-I;;CRISPR/Cas9;;MDCK cell line;;gene knockout;;H9N2 subtype influenza virus
摘 要：
The RIG-I knockout MDCK cell line was constructed using CRISPR/Cas9 technology and detected its biological function. Two targeting vectors(sgRNA) targeting RIG-I gene were designed and constructed, and MDCK cells were co-transfected with pCAG-Cas9-EGFP expression vector. The cell lines were detected through genotype detection,gene sequencing and western blot and infected by H9 N2 subtype avian influenza virus(AIV), and the mRNA expression levels of MAVS, IRF3, IFN-β, IL-6, Mx1 were detected by real-time PCR. The H9 N2 AIV viral gene copy numbers were also detected by real-time PCR, TCID_(50) was used to determine the level of virus replication.The results showed that two MDCK cells(MDCK-RIG-I~(-/-)) with stable knockout RIG-I gene were obtained, and western blot showed that RIG-I protein was not expressed. The results of real-time PCR showed that the mRNA levels of MAVS, IRF3, IFN-β, IL-6 and Mx1 of MDCK-RIG-I~(-/-)cell line decreased significantly after virus infection, indicating the Type Ⅰ interference signal pathway that mediated by RIG-I was blocked after RIG-I gene knockout. The results of viral gene copy number and TCID_(50) showed that the difference was up to 2.16 times and 3.19 times of wild-type MDCK, indicating that the replication of influenza virus was increased after RIG-I gene knockout. In this study, a MDCK cell line of RIG-I gene knockout was established by CRISPR/Cas9 technology, laid a foundation for studying the natural immune response mechanism of RIG-I. This technical method and strategy could provide candidate cell lines for influenza vaccine production and industrial upgrading.
