酵母双杂交筛选肝细胞中乙型肝炎病毒表面抗原中蛋白结合蛋白基因

Screening proteins in hepatocytes interacting with the middle surface protein of hepatitis B virus using the yeast-two hybrid technique

目的筛选并克隆人肝细胞中与HBsAg中蛋白（MHBs）相互作用肝细胞蛋白的基因，探讨MHBs的生物学功能。方法应用PCR法以HBVadr亚型全序质粒A7为模板扩增MHBs基因，克隆到pGEM-T载体中扩增并测序鉴定，EcoRI和BamHI双酶切后回收连接到酵母表达载体pGBKT-7中并应用酵母双杂交系统3，构建MHBs诱饵质粒并转化酵母AH109，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-半乳糖苷酸上进行双重筛选阳性菌落，提取阳性酵母菌落的质粒转化大肠杆菌氨苄青霉素-LB平板上，选择并测序结果在GenBank中进行生物信息学分析。结果构建MHBs酵细胞表达载体，配合后筛选出既能在四缺培养基（SD／-Trp-Leu-Ade-His）上培养也能在铺有X-α-半乳糖苷酸的四缺培养基上生长，并变成蓝色的真阳性菌落2个，其中一个克隆为人醛缩酶B、另一个克隆为未知功能基因，新基因命名为MBPI。结论扩增出MHBs基因，用酵母双杂交技术筛选出2个与MHBs相互作用的肝细胞结合蛋白编码基因，为阐明MHBs的生物学功能及HBV损害肝细胞、致癌等方面的作用提供了新线索。

Objective To screen proteins in hepatocytes interacting with hepatitis B virus surface antigen middle protein （MHBs） with yeast-two hybrid technique for studying the biological functions of MHBs. Methods Polymerase chain reaction （PCR） was used to amplify the gene of MHBs from the plasmid A7 containing the whole fragment of adr subtype of HBV and the PCR product was cloned into pGEM-T vector and then evaluated by sequencing. The gene of MHBs was cut by EcoRI and BamH I from pGEM-T vector and then cloned into the yeast expression plasmid pGBKT7. MHBs bait plasmid was constructed by ligating MHBs gene with yeast expression vector pGBKT7 with yeast-two hybrid system 3 and then was transformed into yeast AHI09 （a type）. The transformed yeast cells were mated with yeast Y187 （ a type） containing liver cDNA library plasmid in 2 x YPDA medium. Diploid yeast cells were plated on synthetic dropout nutrient medium （SD/-Trp-Leu-His-Ade） and synthetic dropout nutrient medium （SD/-Trp-Leu-His- Ade） containing X- α -gal for selecting and screening. After extracting and sequencing the plasmid from true positive blue colonies, the results were analyzed by bioinformatics. Results A pGBKT7- MHBs yeast expressed vector was successfully constructed. Two colonies were sequenced. One colony was Homo sapiens aldolase B fructose-bisphosphate, the other was a new gene with unknown function, which was named MHBs- binding protein I. Conclusion MHBs gene was successfully cloned. Two genes of MHBs interacting proteins in hepatocytes were obtained by yeast-two hybrid system 3. Our results brought some new clues for studying the biological functions of MHBs and the mechanisms of HBV carcinogenesis.