目的探讨丙型肝炎病毒(HCV)core、NS3、NS5A对内源性IFN-β表达的影响及其调控机制.方法将HCV core、NS3、NS5A表达载体pcDNA3.1/myc-His-core/NS3/NS5A转染至HepG2细胞,验证蛋白表达之后,采用实时荧光定量PCR和Western blot及ELISA方法观察3种蛋白对IFN-βmRNA及蛋白水平表达的影响.构建IFN-β全长启动子报告基因表达载体,借助双萤虫素酶活性检测,探讨HCV core、NS3、NS5A对IFN-β转录水平的调控机制.结果 pcDNA3.1/myc-His-core/NS3/NS5A在HepG2细胞中成功表达,与转染pcDNA3.1/myc-His空载体相比,pcDNA3.1/myc-His-NS3/NS5A过表达时,在mRNA及蛋白水平均能抑制HepG2细胞内IFN-β的表达,与转染空载体的对照组相比,差异有统计学意义(P <0.05).双萤虫素酶活性检测显示,转染IFN-β全长启动子报告基因表达质粒后,与对照组相比,双萤虫素酶活性降低,差异有统计学意义(P<0.05).pcDNA3.1/myc-His-core过表达时对IFN-β的表达无明显影响.结论HCV NS3/NS5A在mRNA及蛋白水平能抑制IFN-β表达,并通过其转录水平影响IFN-β表达, core对IFN-β的表达无明显影响.其具体调控机制有待进一步研究.%Objective To investigate the effects and regulation mechanism of HCV core/NS3/NS5A on expression of IFN-βin HepG2 cells. Methods HCV core/NS3/NS5A proteins were cloned (pcDNA3.1/myc-His-core/NS3/NS5A) and expressed in HepG2 cells, then the expression level of IFN-β was detected by qRT-PCR, Western blot hybridization and ELISA. IFN-β promoter reporter vector (PGL4.10-IFN-β-P) was constructed and used for regulation mechanism study by luciferase assay. Results HCV core/NS3/NS5A proteins were successfully expressed in HepG2 cells post-transfection. Both the mRNA level and protein expression of IFN-βwere significantly decreased (P<0.05) in the presence of NS3/NS5A protein. Luciferase assay revealed that NS3/NS5A proteins downregulated IFN-βpromoter activity (P < 0.05). Meanwhile, HCV core protein had no significant effect on IFN-βexpression. Conclusions Overexpression of NS3/NS5A could inhibit the expression of IFN-βby downregulating IFN-βpromoter activity.
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