Chen Guo-feng, Wang Lin, Cheng Jun, Zhang Ling-xia, Li Li, Zhang Jian, Shao Qing, Ji Dong
The 4th Department, The No 302 Hospital of The People's Liberation Army, Beijing 100039, China.
Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi. 2005 Mar;19(1):84-6.
To screen and clone the genes of protein interacting with the N-terminal protein (TP) of hepatitis B virus DNA polymerase.
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 x YPDA medium. Diploid yeast was plated on synthetic dropout medium (SD/-Trp-Leu-His-Ade) and that containing X-alpha-GAL for selecting two times and screening. Plasmids were extracted from blue colonies, and sequence analysis was performed by bioinformatics.
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.
Genes encoding TP interacting proteins in hepatocytes were successfully cloned and the results suggest that TP has a wide variety of biological functions.
筛选并克隆与乙型肝炎病毒DNA聚合酶N端蛋白（TP）相互作用的蛋白质基因。
采用聚合酶链反应（PCR）扩增TP，利用酵母双杂交系统3构建TP诱饵质粒，然后转化至酵母AH109。将转化后的酵母与含有肝脏cDNA文库质粒的酵母Y187在2×YPDA培养基中进行交配。将二倍体酵母接种在合成缺陷培养基（SD/-Trp-Leu-His-Ade）和含有X-α-GAL的培养基上进行两次筛选。从蓝色菌落中提取质粒，并通过生物信息学进行序列分析。
获得47个克隆，这些克隆包括人P36956固醇调节元件结合蛋白-1、RNA聚合酶II亚基hsRPB7 mRNA、去唾液酸糖蛋白受体2转录变体3、铜蓝蛋白（铁氧化酶）、跨膜4超家族成员2以及19个假定蛋白等。
成功克隆了肝细胞中编码与TP相互作用蛋白的基因，结果表明TP具有多种生物学功能。
