酵母双杂交技术筛选乙型肝炎病毒DNA聚合酶TP结合蛋白的研究  

Screening and cloning of the genes of protein interacting with the N-terminal protein of hepatitis B virus DNA polymerase by yeast-two hybrid technique

作　　者：陈国凤[1] 王琳 成军 张玲霞 李力[1] 张健[1] 邵清[1] 纪冬 

机构地区：[1]解放军第三○二医院感染四科,北京100039 [2]传染病研究所基因治疗研究中心 [3]传染病研究所专家组

出　　处：《中华实验和临床病毒学杂志》2005年第1期84-86,共3页Chinese Journal of Experimental and Clinical Virology

基　　金：国家自然科学基金攻关项目 (C0 3 0 114 0 2 ;C3 0 0 70 689) ;军队"九;五"科技攻关项目 (98D0 63 ) ;军队回国留学人员启动基金项目 (98H0 3 8) ;军队"十;五"科技攻关青年基金项目 (0 1Q13 8) ;军队"十;五"科技攻关面上项目 (0 1MB13 5 )

摘　　要：目的　筛选与乙型肝炎病毒 (HBV)DNA聚合酶-N末端蛋白 (TP)相互结合的肝细胞蛋白 ,进一步探讨TP的生物学功能。方法　用聚合酶链反应 (PCR)技术扩增TP片段 ,连接入酵母表达载体pGBKT7中构建诱饵质粒 ,转化酵母细胞AH 10 9并在其内表达 ,然后与转化了人肝cDNA文库质粒pACT2的酵母细胞Y187进行配合 ,在营养缺陷型培养基和X-α-半乳糖苷酶 (X-α-gel)上进行双重筛选阳性菌落 ,DNA序列测定并进行生物信息学分析。结果　成功获得了 4 7个与TP特异性结合的阳性克隆 ,包括人类固醇调节元件结合蛋白 1(SREBP1)、RNA聚合酶Ⅱ亚单位 (hsRPB7)、血浆铜蓝蛋白(CP)等 2 1种已知功能蛋白质基因和 19个假设蛋白基因。结论　HBVDNA聚合酶-TP可以与某些已知和未知功能蛋白相互结合 。Objective To screen and clone the genes of protein interacting with the N-terminal protein (TP) of hepatitis B virus DNA polymerase. Methods TP was amplified by polymerase chain reaction(PCR) and TP bait plasmid was constructed by using yeast two-hybrid system 3, then transformed into yeast AH 109. The transformed yeast was mated with yeast Y187 containing liver cDNA library plasmid in 2×YPDA medium. Diploid yeast was plated on synthetic dropout medium(SD/-Trp-Leu-His-Ade) and that containing X-α-GAL for selecting two times and screening. Plasmids were extracted from blue colonies, and sequence analysis was performed by bioinformatics. Results Forty-seven clones were obtained, these clones included human P36956 sterol regulatory element binding protein-1, RNA polymerase Ⅱ subunit hsRPB7 mRNA, asialoglycoprotein receptor 2, transcript variant 3, ceruloplasmin (ferroxidase), transmembrane 4 superfamily member 2 and 19 of the hypothetical proteins and so on. Conclusion Genes encoding TP interacting proteins in hepatocytes were successfully cloned and the results suggest that TP has a wide variety of biological functions.

关 键 词：TP 乙型肝炎病毒DNA 结合蛋白 酵母双杂交技术 筛选 质粒 血浆铜蓝蛋白 DNA聚合酶 阳性克隆 RNA聚合酶Ⅱ 

分 类 号：R346[医药卫生—基础医学]