基于CRISPR/Cas9技术的MAVS和NFκB1基因敲除MDCK细胞系的构建及初步鉴定  

Construction and preliminary identification of MAVS or NFκB1 gene knockout in MDCK cells using CRISPR/Cas9

作　　者：田棣[1] 刘祎[1] 陈海丽[1] 杨春忆 王蔚 易志刚 张万菊[1] Tian Di;Liu Yi;Chen Haili;Yang Chunyi;Wang Wei;Yi Zhigang;Zhang Wanju(Shanghai Public Health Clinical Center,Shanghai 201508,China)

机构地区：[1]上海市公共卫生临床中心,201508

出　　处：《中华实验和临床病毒学杂志》2020年第2期197-201,共5页Chinese Journal of Experimental and Clinical Virology

基　　金：上海市卫生健康委员会科研课题计划(201540147);国家科技重大专项"艾滋病和病毒性肝炎等重大传染病防治"(2017ZX0103009)。

摘　　要：目的构建线粒体抗病毒信号(mitochondrial antiviral signaling,MAVS)和核因子κB1(nuclear factor kappa B1,NFκB1)基因敲除的犬肾细胞系(Madin-Daby canine kidney cells,MDCK),对细胞系的生长特性、流感病毒(influenza virus,Flu)的增殖特性进行初步鉴定。方法采用CRISPR/Cas9技术,将MDCK细胞的MAVS和NFκB1基因敲除,获得稳定的敲除细胞系。接种Flu并检测培养上清中病毒的血凝(hemagglutinination,HA)滴度和TCID50滴度,与野生MDCK细胞进行比较。结果获得2株稳定敲除的MDCK细胞系MDCK-MAVS-和MDCK-NFκB1-,与野生MDCK细胞同样为贴壁的上皮样细胞。接种Flu后,2株敲除细胞系测定的病毒HA滴度与野生细胞相比无明显差异,TCID50分别比野生MDCK细胞系提高了9.5倍和10倍,其差异有统计学意义(P=0.0316)。结论本研究构建的2株基因敲除细胞系MDCK-MAVS-和MDCK-NFκB1-能够提高Flu的感染力,为Flu的分离培养提供了新的候选细胞系。Objective To construct mitochondrial antiviral signaling(MAVS)gene or Nuclear factor kappa B1(NFκB1)gene knockout Madin-Darby canine kidney(MDCK)cells,and identify the cell growth and proliferation characteristics of influenza virus(Flu)in these cells.Methods MAVS or NFκB1 knockout MDCK cells were established using clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated 9(Cas9)technique.Flu was inoculated into these cells,then the hemagglutination(HA)titers and TCID50 were determined and compared with the original MDCK cells.Results MDCK cells knocked out of MAVS(MDCK-MAVS-)or NFκB1(MDCK-NFκB1-)were obtained stably.The two cells were both adherent epithelioid cells as well as wild type MDCK cells.The HA titers showed no obvious difference among these cells after inoculating Flu.Nevertheless,the TCID50 titers were respectively increased 9.5 times in MDCK-MAVS-cell culture and 10 times in MDCK-NFκB1-cell culture,compared to the wild type MDCK cells.Conclusions The infectivities of Flu were both increased in MDCK-MAVS-cells and MDCK-NFκB1-cells,illustrating their potential in Flu culture.

关 键 词：CRISPR/Cas9 犬肾细胞系 线粒体抗病毒信号 核因子 基因敲除 

分 类 号：R[医药卫生]