酵母双杂交技术筛选克隆HCV核心蛋白结合蛋白基因1  

Screening and Cloning of gene ofhepatocyte protein 1 interacting with HCV core protein

作　　者：李克[1] 王琳[1] 成军[1] 张玲霞[1] 段惠娟[1] 陆荫英[1] 杨继珍[1] 刘妍[1] 洪源[1] 夏小兵[1] 王刚[1] 董菁[1] 李莉[1] 钟彦伟[1] 陈菊梅[1] 

机构地区：[1]中国人民解放军第302医院传染病研究所基因治疗研究中心,北京市100039

出　　处：《世界华人消化杂志》2001年第12期1379-1383,共5页World Chinese Journal of Digestology

基　　金：国家自然科学基金No.39970674

摘　　要：目的克隆与丙型肝炎病毒(HCV)核心蛋白结合的肝细胞中的未知蛋白基因。方法应用酵母双杂交系统3.构建HCV核心蛋白诱饵质粒,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。筛选出30个阳性克隆,对既能在4缺营养缺陷培养基上生长(SD/-Trp-Leu-Ade-His)又能在涂有X-α-半乳糖(X-α-gal)的四缺培养平皿上长出蓝色菌落,提取此酵母克隆的质粒转化大肠杆菌后进行测序,进行生物信息学分析.将HCV核心蛋白基因分为大、中、小三段,与所克隆的未知功能基因回交,以证实其相互作用的部位。结果分析的结果显示,其中1号克隆的测序结果与GenBank中的1个未知功能序列有99％的同源性,初步证明了此基因与HCV核心蛋白有结合活性(GenBank号:AY032593).HCV核心蛋白的全长、2/3,1/3三段,回交显示三段均能在酵母中与此未知基因具有结合作用。结论 HCV核心蛋白结合蛋白肝细胞基因克隆成功,核心蛋白与此未知基因的作用部位应是氨基端.此项研究结果,为以后研究此基因在HCV致病机制以及在肝细胞中的生理功能提供了线索。AIM To clone the unknown gene of hepatocyte protein interacting with hepatitis C virus core protein. METHODS Using the yeast-two hybrid system 3, we constructed bait plasmid of hepatitis C virus core. After it was proved that hepatitis C virus core protein could firmly expressed in AH109 yeast strain, we performed yeast-two hybrid by mating AH109 with Y187 that transformed with liver cDNA library plasmids pACT2 and then plated on quadrople dropout (QDO) medium and assayed for X-α-gal activity. The genes from yeast colonies could grow on QDO and had α-gal activity. We also divided the gene of HCV core into 3 parts, core (L), 2/3 core (M) and 1/3 core (S) fragments, three fragments was crossed back with the gene we had cloned. RESULTS Among the 30 positive colonies, we blasted the gene of the first colony named human hepatitis C virus binding protein 1 (Hu Hcbpl) with GenBank, Hu Hcbpl has as much as 98% homologues with two cDNA which had no known functions. We proved that Hu Hcbpl could interacted with hepatitis C virus core protein and all three parts L, M, S also could interact with Hu Hcbpl. CONCLUSIONS Hepatitis C virus core binding protein (Hu Hcbpl)was successfully cloned and identified. The part that is interacted with Hu Hcbpl is supposedly located in amino end of HCV core. The study paved the way for the investigation of HCV pathogenesis and physiological function of the Hu Hcbpl.

关 键 词：C型肝炎样病毒属 遗传学 病毒蛋白质类 遗传学 病毒基因 分子克隆 遗传学技术 

分 类 号：R373.21[医药卫生—病原生物学]