论文部分内容阅读
目的探讨丙型肝炎病毒(HCV)NS3对干扰素-λⅠ(IFN-λⅠ/IL-29)表达的影响及其调控机制。方法将HCV NS3表达载体pcDNA3.1/myc-His-NS3转染至HepG2细胞,验证HCV NS3蛋白表达之后,采用实时荧光定量PCR、Westernblot和ELISA法观察HCV NS3对IFN-λⅠmRNA及其蛋白表达水平的影响。构建IFN-λⅠ全长启动子报告基因表达载体和3’-非翻译区(3’-UTR)报告基因表达载体,借助双萤光素酶活性检测,探索HCV NS3对IFN-λⅠ转录水平的调控机制。结果 pcDNA3.1/myc-His-NS3在HepG2细胞中成功表达,与转染pcDNA3.1/myc-His空载体相比,pcDNA 3.1/myc-His-NS3过表达时在mRNA和蛋白水平均能抑制HepG2细胞内IFN-λⅠ的表达,差异具有统计学意义(P<0.05)。双萤光素酶活性检测结果显示,与对照组相比,转染IFN-λⅠ全长启动子和3’-非翻译区报告基因表达质粒后,双萤光素酶活性变化无统计学意义(P>0.05)。结论 HCV NS3在mRNA及蛋白水平能抑制IFN-λⅠ的表达,但其转录水平不受影响,具体调控机制尚有待进一步研究。
Objective To investigate the effect of hepatitis C virus (HCV) NS3 on the expression of interferon-λⅠ (IFN-λⅠ / IL-29) and its regulatory mechanism. Methods HCV NS3 expression vector pcDNA3.1 / myc-His-NS3 was transfected into HepG2 cells to verify the HCV NS3 protein expression, real-time fluorescent quantitative PCR, Western blot and ELISA were used to observe the effect of HCV NS3 on IFN-λⅠmRNA and its protein expression Impact. The full-length promoter of IFN-λⅠ and the 3’-UTR region of the 3’-untranslated region (3’-UTR) reporter gene were constructed to detect the transcription of IFN-λⅠ by dual luciferase assay mechanism. Results pcDNA3.1 / myc-His-NS3 was successfully expressed in HepG2 cells. Compared with pcDNA3.1 / myc-His empty vector, pcDNA3.1 / myc-His-NS3 overexpressed at both mRNA and protein levels Inhibition of HepG2 cells IFN-λ Ⅰ expression, the difference was statistically significant (P <0.05). The results of luciferase assay showed that compared with the control group, the luciferase activity of transfection with IFN-λⅠ full-length promoter and 3’-untranslated region gene expression plasmid had no statistical significance P> 0.05). Conclusion HCV NS3 can inhibit the expression of IFN-λⅠ at mRNA and protein levels, but the transcription level of HCV NS3 is not affected. The exact regulatory mechanism remains to be further studied.