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目的研究丙型肝炎病毒编码的蛋白对RNA干扰的抑制作用,筛选出有作用的RNA干扰抑制因子。方法构建丙型肝炎病毒不同蛋白质的真核表达质粒,加入萤火虫荧光素酶基因的RNA干扰体系,共转染HeLa细胞,检测萤火虫荧光素酶活性以观察RNA干扰效果的变化,筛选出有作用的RNA干扰抑制因子。在针对内源性GFP的RNA干扰体系中进一步确证该种抑制作用。利用共转染方法观察该蛋白抑制RNA干扰的剂量效应关系、在不同细胞系中的作用以及对siRNA介导的RNA干扰是否有相同的拮抗作用。结果加入核心蛋白后,萤火虫荧光素酶活性恢复到80%,与空白对照比较有显著性差异(P<0.05)。在绿色荧光蛋白RNA干扰体系中,核心蛋白使绿色荧光蛋白的表达强度明显增加。该种对抗RNA干扰的作用在HepG2、HEK293和HeLa细胞中均存在,且呈剂量依赖关系。在siRNA介导的RNA干扰体系中,加入核心蛋白后萤火虫荧光素酶活性无显著性变化(P>0.05)。结论核心蛋白能够对抗shRNA介导的RNA干扰作用,但不能对抗siRNA介导的RNA干扰作用,这种作用具有普遍性。核心蛋白对抗RNA干扰的作用可能有利于提高丙型肝炎病毒在宿主细胞内的生存能力并导致持续感染,亦可能在丙型肝炎病毒致病机制中发挥重要作用。
Objective To study the inhibitory effect of HCV-encoded protein on RNA interference, and to screen for a useful RNA interference inhibitor. Methods The eukaryotic expression plasmids of different proteins of hepatitis C virus were constructed. The RNA interference system of firefly luciferase gene was added and co-transfected into HeLa cells. The firefly luciferase activity was detected to observe the change of RNA interference effect. RNA interference inhibitors. This inhibition was further confirmed in RNA interference systems targeting endogenous GFP. The co-transfection method was used to observe the dose-effect relationship of this protein in inhibiting RNA interference, its role in different cell lines and whether it has the same antagonism to siRNA-mediated RNA interference. Results After adding the core protein, the firefly luciferase activity returned to 80%, which was significantly different from that of the blank control (P <0.05). In the GFP RNA interference system, the core protein significantly increases the expression intensity of green fluorescent protein. The antagonism of RNA interference in HepG2, HEK293 and HeLa cells are present in a dose-dependent manner. There was no significant change of firefly luciferase activity in siRNA-mediated RNA interference system (P> 0.05) after addition of core protein. Conclusion The core protein is able to antagonize shRNA-mediated RNA interference but not siRNA-mediated RNA interference. This effect is universal. The role of core protein against RNA interference may be beneficial to improve the viability of hepatitis C virus in host cells and lead to persistent infection. It may also play an important role in the pathogenesis of hepatitis C virus.