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

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.

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

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-a-gal activity. Nineteen yeast colonies which grew on QDO and had a-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.