干扰素刺激基因12a对寨卡病毒复制的影响及其作用机制  

Effect of interferon-stimulated gene 12a on Zika virus replication and the underlying mechanism

作　　者：朱安静 李爽 李玉佳[1] 李世林[1] 段晓琼[1] 杨春晖[1] 徐敏[1] 陈利民[1] ZHU Anjing;LI Shuang;LI Yujia;LI Shilin;DUAN Xiaoqiong;YANG Chunhui;XU Min;CHEN Limin(Institute of Blood Transfusion,Chines Academy of Medical Science,Peking Union Medical College,Provincial Key Laboratory for Transfusion-Transmitted Diseases of Sichuan Province,Chengdu,610052 China)

机构地区：[1]中国医学科学院,北京协和医学院输血研究所,四川省输血传播疾病医学重点实验室,四川成都610052

出　　处：《中国输血杂志》2020年第5期446-452,共7页Chinese Journal of Blood Transfusion

基　　金：国家重点研发计划项目(2018YFE0107500)。

摘　　要：目的探究干扰素刺激基因(ISG)12a对寨卡病毒(ZIKV)复制的影响和作用机制。方法通过质粒转染(2μg ISG12a)或者siRNA(20μmo/Ll)沉默分别上调或者抑制A549细胞(细胞密度2×10^5/mL)中ISG12a的表达,再用ZIKV(MOI=1)感染A549细胞,48 h后收样,采用RT-qPCR以及Western blot等方法检测ZIKV的RNA的复制水平及宿主抗病毒先天免疫相关基因表达水平;将1μg含有干扰素刺激应答元件(ISRE)的萤火虫荧光报告质粒(ISRE-luc)和2 ng海肾荧光质粒(pRL-TK)与1μg的ISG12a质粒共转入2×10^5/mL的A549细胞中24 h,用ZIKV(MOI=1)感染A549细胞24 h,用终浓度为100 IU/mL IFNβ再处理细胞24 h后收样,用双荧光报告基因试验检测ISRE活性;在2×10^5/mL的A549细胞中转染2μg ISG12a质粒,24 h后用ZIKV(MOI=1)感染A549细胞24 h,用终浓度为100 IU/mL IFNβ再处理细胞24 h后收样,采用RT-qPCR检测各组(n=3)的ISG12a和ZIKV RNA的表达。结果过表达ISG12a或者沉默ISG12a使得实验组的ZIKV RNA相对于空载组的值为:0.438 8±0.011 3、1.884 0±0.176 7(P<0.01),使IFNβ及MDA5、RIG-I、TLR3、IFIT1、ISG15、MxA各组(n=3)的相对值为:1.925 2±0.294 0、3.847 1±0.245 3、3.535 2±0.461 3、3.584 0±0.243 2、17.304 3±0.963 7、19.393 5±5.074 6、21.909 3±1.941 6(P<0.01或<0.05);在感染ZIKV的情况下,过表达ISG12a后,用IFNβ处理细胞,与空载组相比,实验组的ZIKV RNA的相对值为:218.723 3±0.329 9、2.330 6±0.080 6(P<0.01),与不加IFNβ处理的实验组相比,加干扰素处理的实验组的ZIKV RNA的相对值为:1.312 9±0.010 4(P<0.05)。结论 ISG12a通过促进内源性IFNβ的产生和激活Ⅰ型干扰素信号通路从而抑制ZIKV的复制,并增强外源性IFNβ抗ZIKV活性。Objective To study the role of interferon stimulated gene 12 a(ISG12 a) in Zika virus(ZIKV) replication and the underlying mechanism. Methods ISG12 a was upregulated or down-regulated by plasmid transfection(2 μg) or RNAi(20 μmol/L), respectively. 24 h after transfection, inoculating ZIKV with MOI=1, and after 48 h of treatment, RT-qPCR and western blot were used to determine the replication of ZIKV RNA, as well as the expression of host anti-virus innate immunity-related genes. 1 μg firefly fluorescent reporter plasmid(ISRE-luc) containing interferon-stimulated response element(ISRE) and 2 ng Renilla fluorescent reporter plasmid(pRL-TK) were co-transfected with 1 μg of ISG12 a into A549 of 2×10^5/mL, 24 h after transfection, inoculation ZIKV with MOI=1, 24 h after inoculation,treatment cells with IFNβ(200 IU/mL), after 24 h of treatment, detecting the expression of interferon-stimulated response element(ISRE) activity(dual-luciferase reporter assay). 2 μg ISG12 a was transfected, 24 h after transfection,inoculation ZIKV with MOI=1, after 24 h of inoculation, treatment cells with IFNβ(200 IU/mL). After 24 h of treatment, ZIKV replication was assessed by RT-qPCR to determine the effect of ISG12 a on anti-ZIKV activity of IFNβ. Results Overexpression of ISG12 a or knockdown of ISG12 a expression makes the ISG12 a groups′ ZIKV RNA relate to the mock-vehicle groups expression: 0.438 8±0.011 3,1.884 0±0.176 7(P<0.01), and making the relative expression levels of IFNβ,MDA5,RIG-I,TLR3,IFIT1,ISG15,MxA are 1.925 2±0.294 0,3.847 1±0.245 3,3.535 2±0.461 3,3.584 0±0.243 2,17.304 3±0.963 7,19.393 5±5.074 6,21.909 3±1.941 6(P<0.01 or P<0.05). In the case of ZIKV infection, after overexpressing ISG12 a, cells were treated with IFNβ. Compared to the mock-vehicle groups, the relative expression levels of ZIKV RNA in the experimental groups were: 218.723 3±0.329 9,2.330 6±0.080 6(P<0.01), and compared to the experimental groups without IFNβ treatment, the relative expression levels of the experimental grou

关 键 词：寨卡病毒 干扰素刺激基因12a 干扰素Β Janus酪氨酸激酶/信号传导转录激活因子信号通路 

分 类 号：Q5525[生物学—生物化学] R373.3[医药卫生—病原生物学]