肠道病毒-D68 2A蛋白对抗病毒IFN-Ⅰ通路影响的研究  

Enterovirus D68 protease 2A affects anti-viral interferon type Ⅰ pathway

作　　者：郑惠文[1] 杨智尧 杨泽宁[1] 宋杰[1] 黄星 李楠 丁丽莎 李恒 李洪哲[1] 郭磊[1] 储曼曼 施海晶 刘龙丁[1] Zheng Huiwen;Yang Zhiyao;Yang Zening;Song Jie;Huang Xing;Li Nan;Ding Lisha;Li Heng;Li Hongzhe;Guo Lei;Chu Manman;Shi Haijing;Liu Longding(Institute of Medical Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Kunming 650118, China;University of Iowa, Iowa 52241, USA)

机构地区：[1]中国医学科学院&北京协和医学院医学生物学研究所,昆明650118 [2]美国爱荷华大学,52241

出　　处：《中华微生物学和免疫学杂志》2019年第6期401-409,共9页Chinese Journal of Microbiology and Immunology

基　　金：中国医学科学院医学与健康科技创新工程(2017-I2M-2-006);国家自然科学基金(31570900).

摘　　要：目的观察肠道病毒(EV)-D68蛋白2A在EV-D68病毒感染293T细胞过程中对抗病毒IFN-Ⅰ通路的影响。方法免疫印迹法(Western blot)检测重组2A蛋白、IFN-α因子及信号传导及转录激活因子1(STAT1)的蛋白表达水平;免疫荧光技术(Immunofluorescence,IF)检测细胞内不同时间点EV-D68病毒VP1与2A的表达;通过观察细胞病变效应(cytopathic effect,CPE),计算细胞培养半数感染量(50% cell culture infective dose,CCID50)来检测病毒感染性滴度;利用实时荧光定量PCR技术(real-time PCR,RT-PCR)检测IFN-Ⅰ干扰素产生相关基因的表达。结果成功构建pCLIPf-2A质粒,在转染后的24 h,重组蛋白2A有较好的表达。病毒滴度结果表明,2A蛋白在感染后期可以有效促进EV-D68病毒的增殖复制。IFN-α干扰素检测结果表明,EV-D68 2A可以刺激IFN-α的表达。IFN-Ⅰ干扰素相关基因检测表明,EV-D68+2A组、2A组及EV-D68对照组的IFN-Ⅰ干扰素产生相关基因的IFN-α mRNA水平均有不同程度的上调或下调,但是三者诱导相关基因表达上调、下调的趋势不同。对IFN-Ⅰ干扰素通路下游蛋白STAT1检测结果表明,各组均能有效诱导STAT1蛋白的表达。结论EV-D68 2A可以促进抗病毒IFN-Ⅰ通路相关基因及下游蛋白的应答,病毒感染后期,2A可以促进病毒较快增殖。Objective To analyze how enterovirus D68(EV-D68)protease 2A affects the anti-viral interferon type Ⅰ(IFN-Ⅰ)pathway in 293T cells following infection. Methods Western blot was used to detect the expression of recombinant protease 2A, IFN-α and signal transducers and activators of transcription 1(STAT1)at protein level. Expression of EV-D68 viral protein(VP1)and protease 2A was analyzed by immunofluorescence at different time points. Cytopathic effects were recorded to calculate 50% cell culture infective dose(CCID50). Expression of the genes involved in the anti-viral IFN-Ⅰ pathway was measured by real-time PCR(RT-PCR). Results The recombinant plasmid pCLIPf-2A was successfully constructed and the expression of recombinant protease 2A could be detected by Western blot 24 h after transfection. The recombinant protease 2A promoted the proliferation of EV-D68 at the late stage of infection and induced the production of IFN-α. Expression of the genes involved in the anti-viral IFN-Ⅰ pathway at mRNA level was up- or down-regulated to different degrees with various trends in different groups following infection. Expression of STAT1 was enhanced in all groups. Conclusions EV-D68 protease 2A promoted the activation of anti-viral IFN-Ⅰpathway in response to viral infection and enhanced the proliferation of virus at the late stage of infection.

关 键 词：EV-D68病毒感染 2A蛋白 抗病毒IFN-Ⅰ通路 

分 类 号：R373[医药卫生—病原生物学]