论文部分内容阅读
目的探讨核心蛋白与Dicer的相互作用及其对Dicer功能的影响。方法构建丙型肝炎病毒核心蛋白真核表达质粒,采用免疫荧光染色和Western blot检测核心蛋白的表达,Western blot检测核心蛋白对细胞内Dicer表达的影响,免疫共沉淀技术检测真核细胞内核心蛋白与Dicer的相互作用。体外转录合成dsRNA,检测核心蛋白对Dicer切割dsRNA作用的影响。结果在细胞内,核心蛋白能够明显恢复被shRNA抑制的虫荧光素酶基因的表达(P<0.05),而对siRNA引发的RNAi无拮抗作用。在真核细胞内,加入核心蛋白后,Dicer的表达量无明显改变,并能检测到两者相互结合。在体外抑制实验中,重组表达的Dicer能切割dsRNA产生siRNA;核心蛋白加入后,dsRNA的切割受到抑制。结论核心蛋白在真核细胞内可以结合Dicer,并通过抑制Dicer对dsRNA的切割作用,抑制RNA干扰作用。
Objective To explore the interaction between core protein and Dicer and its effect on Dicer function. Methods The eukaryotic expression plasmid of hepatitis C virus core protein was constructed. The expression of core protein was detected by immunofluorescence staining and Western blot. The effect of core protein on the expression of Dicer was detected by Western blot. The co-immunoprecipitation assay was used to detect the expression of core protein Interaction with Dicer. In vitro transcriptional dsRNA synthesis, detection of core protein Dicer cleavage of dsRNA effect. Results In the cells, the core protein could obviously restore the expression of luciferase gene inhibited by shRNA (P <0.05), but not the RNAi induced by siRNA. In eukaryotic cells, after addition of the core protein, the expression of Dicer did not change significantly, and both could be detected. In in vitro inhibition experiments, recombinantly expressed Dicer cleaves dsRNA to produce siRNA; dsRNA cleavage is inhibited after addition of the core protein. Conclusion The core protein can bind Dicer in eukaryotic cells and inhibit RNA interference by inhibiting the cleavage of dsRNA by Dicer.