紫外交联试验筛选结合于丙型肝炎病毒RNA核心区的细胞蛋白

Screening of cellular proteins binding to the core region of hepatitis C virus RNA by ultraviolet cross-linking assay

目的从肝癌细胞系中筛选能够与丙型肝炎病毒(HCV)RNA核心区结合的细胞蛋白质分子。方法采用体外转录法得到HCV RNA核心区片断,从HepG2细胞中提取细胞蛋白质分子;通过核酸-蛋白紫外交联试验筛选HepG2细胞中可与HCV RNA核心区结合的蛋白分子,并用未标记核心区RNA 和载体RNA作为竞争物分析结合的特异性。结果HepG2细胞内有2个约为6.6×104和5.5×104的宿主蛋白分子与HCV RNA的核心区发生结合。未标记核心区RNA可以竞争性抑制地高辛标记的核心区RNA 与细胞蛋白的结合,而载体RNA对此结合无竞争性。结论HepG2细胞内存在着可以与HCV RNA核心区结合的蛋白分子。

Objective To screen cellular proteins binding to the core region of hepatitis C virus （HCV） from human hepatoma cells. Methods Unlabeled and labeled RNA transcripts were prepared by in vitro transcription. Cytoplasmic extracts were prepared from human hepatoma cells HepG2. Ultraviolet （UV） cross-linking was used to screen the cellular proteins that would bind to the core region of HCV. Competition experiment was performed to confirm the specificity of the binding in which excess unlabeled RNA of HCV core region and plasmid RNA were used as competitors. Results Two cellular proteins of 6.6 × 10^4 and 5.5 × 10^4 were found binding to the core region of HCV RNA by UV cross-linking assay. The unlabeled core region of HCV RNA could compete out this binding whereas the unlabeled plasmid RNA could not. Conclusion The cellular proteins from HepG2 cells could bind to the core region of HCV RNA.