Cullin-2基因敲除A549细胞系构建及其对A型流感病毒复制的影响  

Construction of a Cullin-2 Knock-Out A549 Cell Line and Effect on Replication ofInfl uenza A Virus

作　　者：史馨瑾 王俊 刘英楠 陈鸿军[1] SHI Xinjin;WANG jun;LIU Yingnan;CHEN Hongjun(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5期29-35,共7页Chinese Journal of Animal Infectious Diseases

基　　金：国家自然科学基金面上项目资助(31572502)。

摘　　要：CRL复合体是泛素蛋白酶体降解系统中的核心酶,很多病毒会劫持宿主CRL复合体,激活或干扰其底物的降解,创造有利病毒复制的环境。本研究为了探讨CRL2复合体和流感病毒之间的关系,利用CRISPR/Cas9技术构建CRL2复合体骨架蛋白Cullin-2基因稳定敲除A549细胞系,并验证Cullin-2基因敲除对流感复制的影响。设计两条靶向Cullin-2的sgRNA,构建到敲除载体中,包装成慢病毒感染细胞,经过药物筛选和亚克隆纯化后,利用蛋白免疫印迹法和敲除位点测序鉴定,并比较野生型和Cullin-2敲除细胞中AIV的复制效率。结果显示,Cullin-2敲除对流感病毒滴度和病毒蛋白水平影响不显著,但会导致细胞因子转录水平的上调,为CRL复合体与流感病毒致病性机制关系研究提供新的思路。CRL complex is the core enzyme of ubiquitin-proteasome system(UPS).Many viruses can express proteins and interact with host CRL complex during infection,activating or interfering with the degradation of its substrate protein,thus creating an environment benefi cial to virus replication.This study found that in the early stage of Infl uenza virus infection,the transcription level of Cullin-2 was signifi cantly down-regulated.In order to explore the relationship between Cullin-2 and Infl uenza virus,this study used the CRISPR/Cas9 system to construct a Cullin-2 stable knockout A549 cell line and verifi ed its effect on infl uenza virus replication.Two sgRNAs were designed targeting Cullin-2,constructed into Lenti-CRISPRv2 vector,and packaged into lentivirus-infected cells.After drug screening and subcloning screening,the knockout effect was verifi ed by Western blot and sequencing,and the replication effi ciency of AIV in wildtype and Cullin-2 knockout cells was compared.The results showed that Cullin-2 knockout had little effect on the titer and protein level of infl uenza virus,but led to up-regulation of the transcription level of cytokines,providing a new idea for the study of the pathogenicity mechanism of Influenza virus.

关 键 词：A型流感病毒 CRL复合体 Cullin-2 细胞因子 

分 类 号：S852.65[农业科学—基础兽医学]