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-D682A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-αmRNA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-D682A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.

abstractsObjective To analyze how enterovirus D68 (EV-D68) protease 2A affects the anti-vi-ral interferon typeⅠ(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 tran-scription 1 (STAT1) at protein level. Expression of EV-D68 viral protein (VP1) and protease 2A was ana-lyzed 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.