丙型肝炎病毒非结构基因5A反式激活基因4A结合蛋白基因的筛选

Screening and cloning gene of a hepatocyte protein interacting with HCV NS5ATP4A protein

目的 筛选并克隆HCV非结构基因NS5A反式激活基因4剪切体NS5ATP4A的结合蛋白基因，为研究NS5ATP4A的生物学功能提供线索。方法 构建HCV NS5ATP4A蛋白诱饵酵母质粒，转化酵母AH109后与含文库质粒的酵母Y187进行配合，在营养缺陷培养基上进行双杂交筛选。选择既能在4重营养缺陷培养基（SD／-Trp／-Leu／-Ade／-His）上生长，又能在涂有X-α-半乳糖的四缺培养平皿上生长的蓝色菌落，提取此酵母克隆的质粒，转化大肠杆菌后进行测序，并进行生物信息学分析。结果 筛选出7个基因，其中已知功能基因6个，未知功能基因1个，这些基因与RNA合成、蛋白质翻译、细胞周期及肿瘤免疫有关。结论HCV NS5ATP4A结合蛋白基因的成功筛选，提示了HCV NS5ATP4A新的信号转导途径，为HCV致病机制的进一步研究提供了依据。

Objective To screen and clone hepatocyte protein interacting with hepatitis C virus NS5ATP4A protein for studying its biological functions. Methods Bait plasmids of hepatitis C virus NS5ATP4A were constructed. After verifying that hepatitis C virus NS5ATP4A protein could be steadily expressed in AH109 yeast strain, yeast-two hybrid assay was performed by mating AH109 with Y187 which pre-transformed with liver cDNA library plasmids pACT2, and the diploidy yeast cells were plated on quadruple dropout （QDO） medium and assayed for X- α -gal activity. Nineteen yeast colonies which grew on QDO and had α -gal activity were obtained, and then the library plasmids were extracted and sequenced. Results Seven genes were screened out and one of them was a formerly unknown gene. They were associated with RNA synthesis, protein translation, cell cycling and tumor immunity. Conclusion NS5ATP4A binding proteins were successfully screened, which offers new clues for further studying the signal transduction pathway of NS5ATP4A and the pathogenic mechanism of HCV.