ANXA2敲除的A549细胞系的构建及其对H1N1和H9N2流感病毒复制的影响  

ANXA2 knockout A549 cell line construction and effects on the replication of H1N1 and H9N2 influenza viruses

作　　者：刘永宁 马欣傲 吕思莹 李媛 李旭勇[1] 司振书[1] 郭晶[1] 李玉保[1] 刘成[1] LIU Yong-ning;MA Xin-ao;LYU Si-ying;LI Yuan;LI Xu-yong;SI Zhen-shu;GUO Jing;LI Yu-bao;LIU Cheng(College of Agricultural,Liaocheng University,Liaocheng 252000,China)

机构地区：[1]聊城大学农学院,聊城252000

出　　处：《中国人兽共患病学报》2022年第4期291-296,302,共7页Chinese Journal of Zoonoses

基　　金：山东省自然科学基金项目(No.ZR2020MC175);山东省重点研发计划项目(No.2019GNC106082);大学生创新创业训练计划项目(No.CXCY2020Y118,No.CXCY2021287)联合资助。

摘　　要：目的旨在利用CRISPR/Cas9基因编辑技术构建ANXA2敲除的A549细胞,并探索ANXA2敲除对流感病毒复制的影响。方法本研究设计了3对靶向ANXA2基因外显子的特异性sgRNAs,分别将sgRNAs构建到LentiCRISPRv2载体上获得重组质粒,与辅助质粒共转染293T细胞包装成慢病毒后感染A549细胞,通过嘌呤霉素压力及有限稀释法筛选ANXA2基因敲除的单克隆细胞株,用靶基因测序及Western-blot验证ANXA2的敲除效果,并通过CCK-8试验比较ANXA2敲除细胞和野生型A549细胞的细胞活力。再分别用人流感病毒WSN(H1N1)和禽流感病毒SD98(H9N2)感染ANXA2敲除和野生型A549细胞,经TCID_(50)检测ANXA2敲除对流感病毒复制的影响。结果成功获得了ANXA2敲除的A549细胞系,且与野生型A549细胞相比,细胞活力无显著差异;ANXA2敲除后WSN(H1N1)和SD98(H9N2)流感病毒的病毒滴度均升高,其中ANXA2对SD98(H9N2)流感病毒的影响要大于WSN(H1N1)。结论本研究利用CRISPR/Cas9基因编辑技术构建了ANXA2敲除的A549细胞,并发现ANXA2敲除促进了流感病毒的复制,本研究为流感病毒复制和致病机制的研究奠定了基础。The objective of this study was to construct ANXA2 knockout A549 cells by using CRISPR/Cas9 gene editing technology and to explore the effect of ANXA2 knockout on influenza virus replication.To establish ANXA2 knockout cell lines,we designed three specific single guide RNAs(sgRNAs)to target exons of the ANXA2 gene.sgRNAs were inserted into LentiCRISPRv2 to obtain a recombinant plasmid,then transfected into 293T cells with helper plasmids.The resulting lentiviruses were harvested and used to transduce A549 cells.The ANXA2 knockout monoclonal cell line was screened with puromycin through the limiting dilution method.The knockout of ANXA2 in A549 cells was confirmed by target genome sequencing and Western blotting.The cell viability of ANXA2 knockout cells and wild-type cells was verified with CCK-8 experiments.The cells were then infected with human influenza viruses WSN(H1N1)and avian influenza virus SD98(H9N2)to detect the effects of ANXA2 knockout on influenza virus replication,on the basis of the TCID_(50).The ANXA2 knockout A549 cell line was successfully obtained and showed no significant difference in cell viability with respect to that of wild type A549 cells.The viral titers of WSN(H1N1)and SD98(H9N2)influenza virus increased after ANXA2 knockout,and the effect of ANXA2 on SD98(H9N2)influenza virus was greater than that on WSN(H1N1).In conclusion,this study constructed ANXA2 knockout A549 cell lines by using CRISPR/Cas9 gene editing technology and found that ANXA2 knockout promoted influenza virus replication,thus providing a foundation for the study of influenza virus replication and pathogenesis.

关 键 词：CRISPR/Cas9 膜联蛋白A2 基因敲除 流感病毒 

分 类 号：R373.1[医药卫生—病原生物学]