LC3蛋白敲除A549细胞系的构建及其对流感病毒复制的影响  

Construction of a LC3 Protein Knock-Out A549 Cell Line and Evaluation of Its Effect on Influenza Virus Replication

作　　者：陆晨阳 包旦奇 马天馨 牛世奇 张婷 任超超[1] 李雪松[1] 杨健美[1] 滕巧泱[1] 李泽君[1] 刘芹防[1] LU Chenyang;BAO Danqi;MA Tianxin;NIU Shiqi;ZHANG Ting;REN Chaochao;LI Xuesong;YANG Jianmei;TENG Qiaoyang;LI Zejun;LIU Qinfang(Shanghai Veterinary Research Institute,CAAS,Shanghai 200241,China)

机构地区：[1]中国农业科学院上海兽医研究所,上海200241

出　　处：《中国动物传染病学报》2022年第3期7-12,共6页Chinese Journal of Animal Infectious Diseases

基　　金：国家重点研发计划(2017YFD0500806)。

摘　　要：微管相关蛋白1轻链3(LC3)是一种在流感病毒感染过程中被病毒M2蛋白招募至细胞膜处聚集的宿主细胞自噬相关蛋白。为了解其在流感病毒感染过程中行使的功能,利用CRISPR/Cas9技术构建LC3蛋白缺失细胞系,用以观察病毒生长复制的差异。设计sgRNA并插入至核心载体LentiCRISPRv2中,与辅助质粒共同转染293T细胞行慢病毒的包装。包装完成后用以感染A549细胞并进行嘌呤霉素压力筛选,对存活细胞的LC3蛋白含量进行检测,结果显示具有嘌呤抗性的细胞中未检测到LC3蛋白条带,LC3敲除细胞系构建成功。运用核染色法对比敲除细胞和正常细胞的细胞活性,发现缺失LC3蛋白细胞活性小幅度下降。流感病毒感染两种细胞绘制生长曲线,缺少LC3蛋白的条件下病毒复制水平下降。本研究构建的LC3蛋白缺失细胞系能够为将来研究LC3蛋白在细胞自噬与病毒感染过程中发挥功能,以及分子机制的研究提供了良好的细胞模型。Microtubule-associated protein 1 light chain 3(LC3)is one of important marker proteins in autophagy,which is recruited on cytomembrane when host cells get infected with influenza A virus(IAV).In the present study,a LC3 knock-out A549 cell line was constructed using CRISPR/Cas9 system to investigate its effect on IAV replication.First of all,the single guide RNA was designed and inserted into the LentiCRISPRv2 vector.Then,LentiCRISPRv2 was co-transferred with two assist vectors into HEK293T cells to package the Lentivirus.After that,the A549 cells were infected with the Lentivirus and survived positive cells were screened using puromycin filtrate.Then,LC3 protein content of the living cells was detected by Western blot.The result has shown that no LC3 protein expression in the positive cells,indicating successful built up of the LC3 protein knock-out cell line.For comparison the viabilities of the parent A549 and LC3-KO cells,Nucleic Acid Dye method was used for verification.The results showed that the viable LC3-KO cells were lower than A549.Additionally,LC3-KO and A549 cells were infected with IAV to study the virus growth curves.As a result,lack of LC3 caused decrease in viral replication.In summary,the LC3 protein knock-out A549 cell line constructed here might be used as a cell model for the research on LC3 protein function and its role in virus infection.

关 键 词：LC3蛋白 敲除细胞系 流感病毒 感染 

分 类 号：S852.65[农业科学—基础兽医学]