酵母双杂交技术筛选肝细胞中与羧基末端截短型乙型肝炎表面抗原中蛋白MHBs^(t167)蛋白结合蛋白的研究  

Screening and identification of proteins interacting with MHBs^(t167) by yeast-two hybrid technique

作　　者：李志群[1] 马英骥[1] 成军[2] 

机构地区：[1]哈尔滨医科大学第一医院感染科,哈尔滨150001 [2]北京地坛医院传染病研究所

出　　处：《胃肠病学和肝病学杂志》2006年第2期138-140,共3页Chinese Journal of Gastroenterology and Hepatology

摘　　要：目的应用酵母双杂交技术筛选人肝细胞cDNA文库中与羧基末端截短型乙型肝炎病毒表面抗原中蛋白(MHBst167)具有相互作用的肝细胞蛋白,以探讨MHBst167可能的生物学功能。方法用多聚酶链反应(PCR)法扩增MHBst167基因,应用酵母双杂交系统3,连接入酵母表达载体pGBKT7中构建诱饵质粒,转染酵母细胞AH109并在其内表达,然后与转染了人肝cDNA文库质粒pACT2的酵母细胞Y187进行配合,于涂有Xαgal营养缺陷型培养基(SD/TrpLeuHisAde)上进行双重筛选阳性菌落。挑选阳性克隆,提取此酵母克隆的质粒转化DH5α大肠杆菌并经氨苄青霉素抗性筛选,提取单克隆菌落质粒DNA,酶切鉴定后进行测序,然后进行生物信息学分析。结果成功构建MHBst167酵母表达载体pGBKT7MHBst167。筛选出阳性菌落28个,经生物信息学分析,最后从肝细胞cDNA文库中筛选出7个与MHBst167特异性结合作用的克隆。其中包括人类核糖基化因子1、胎儿肝全长cDNA克隆、人类醛缩酶B果糖二磷酸(ALDOB)、补体3(C3)、人类血清扩散因子(生长调节素B)、人类BAC(细菌人工染色体)克隆GS1306C12。结论成功克隆出MHBst167基因并在酵母细胞中表达,应用酵母双杂交技术筛选出7个能与MHBst167蛋白相互作用的肝细胞结合蛋白基因,根据所克隆到的基因,对以后研究MHBst167的生物学功能及乙型肝炎病毒致癌的分子生物学机制奠定了理论基础。Objective To investigate the biological functions of MHBs^t167, we performed yeast-two hybrid to screen proteins interacting with MHBs^t167 from human hepatocyte cDNA library. Methods The MHBs^t167 gene was amplified by polymerase chain reaction （PCR） technique and MHBs^t167 bait plasmid was constructed by ligating the cDNA fragment of MHBs^t167into pG- BKT7 with yeast-two hybrid system 3 , then the yeast expression vector MHBs^t167-pGBKT7 was transfected into yeast AH109 （a type） ,thetransfected yeast were mated with yeast Y187（atype） containing hepatocyte cDNA library plasmid in 2 × YPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium （SD-Trp-Leu-His-Ade） containing x-α-gal for selecting two times and screening. After plasmid DNA extracting, enzyme cutting analysis and sequencing of plasmids from blue colonies, the results underwent sequence analysis by bioinformatics. Results The yeast expression vector MHBs^t167-pGBKT7 was successfully constructed, twenty-eight positive colonies were obtained using yeast-two hybrid technique. After sequence analysis, seven clones were found to have a specially interacting with MHBs^t167protein. Among them, there are colonies such as homo sapiens ADP-ribosylation factor 1, full-length cDNA clone CSODM004YC15 of fetal liver of homo sapiens （human）, homo sapiens ALDOB gene, homo sapiens complement component 3 （C3）, serum spreading factor（somatomedin B）, homo sapiens BAC clone GS1-306C12 from 7. Conclusion MHBs^t167 gene was successful cloned and expressed in yeast ceils. Screened seven protein interacting with MHBs^t167 from human hepatic cDNA libraty by yeast-two hybrid technique, which brought some new clues for studying the biological functions of MHBs^t167 and provided a basis for clarifying the tumor development mechanism of HBV.

关 键 词：乙肝病毒 表面抗原中蛋白 酵母双杂交技术 

分 类 号：R373.2[医药卫生—病原生物学]