应用酵母双杂交技术筛选肝细胞cDNA文库中乙型肝炎病毒前-X蛋白结合蛋白基因

Screening and identification of interacting proteins with pre-X protein of hepatitis B virus in hepatocytes by yeast-two hybrid technique

目的:用酵母双杂交技术筛选肝细胞中与乙型肝炎病毒(HBV)前-X蛋白结合蛋白的编码基因. 方法:用多聚酶链反应(PCR)法扩增HBV基因组中的前-X 基因,连接入酵母表达载体pGBKT-7中构建诱饵质粒,转化酵母细胞AH109并在其内表达,然后与转化了人肝细胞文库质粒的酵母细胞Y187进行配合,在营养缺陷型培养基上进行双重筛选阳性菌落,增菌后提出质粒,转化入大肠杆菌(DH5 α),提出质粒并测序,进行生物信息学分析. 结果:成功克隆出HBV的前-X基因,构建表达载体并在酵母细胞中表达,配合后选出在四重缺陷(SD/-Trp-Leu- Ade-His)培养基及铺有X-α-半乳糖(X-α-gal)的四重缺陷培养基上均能生长并变成蓝色的真阳性菌落19个,其中5 个是人铁蛋白;1个是人的胰岛素样生长因子结合蛋白3 (IGFBP3);1个是人醛缩酶B;1个是人糖基化磷脂酰肌醇锚定结合因子1(GPAA1);1个是人血红素结合蛋白;1个是人Cl酯酶抑制子(Cl-INH);1个是人玻连蛋白;1个是人电压依赖性阴离子通道1(VDAC1);1个是丝氨酸穿膜蛋白酶(hepsin);1个是人梭素;1个是人纤溶酶原(PLG);3个是人假想蛋白;1个是RP11-542M13克隆,位于染色体16. 结论:成功克隆出前-X的结合蛋白,为进一步研究HBV 的前-X蛋白的作用提供了新线索.

AIM: To investigate the biological function of pre-X protein encoded by hepatitis B virus (HBV) genome, and to screen proteins in hepatocytes interacting with pre-X protein by yeast-two hybrid technique. METHODS: The pre-X gene was amplified by polymerase chain reaction (PCR) and pre-X bait plasmid was constructed by using yeast-two hybrid system 3, then the constructed vector was transformed into yeast AH109. The transformed yeast mated with yeast Y187 containing hepatocytes cDNA library plasmid in 2xYPDA medium. Diploid yeast was 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 two times and screening. After extracting and sequencing of plasmid from blue colonies, the results were analyzed by bioinformatics. RESULTS: Nineteen colonies were sequenced, in which five colonies were homo sapiens ferritin, one colonies was homo sapiens insulin-like growth factor binding protein 3 (IGFBP3), one homo sapiens aldolase B, one homo sapiens gene for glycosylphosphatidylinositol anchor attachment 1 (GAA1), one homo sapiens hemopexin, one homo sapiens C1 esterase inhibitor (C1-INH), one homo sapiens vitronectin, one homo sapiens voltage-dependent anion channel 1 (VDAC1), one hepsin (transmembrane protease, serine 1), one homo sapiens spindling, one homo sapiens plasminogen (PLG), three hypothetical proteins, and one homo sapiens chromosome 16 clone RP11-542M13. CONCLUSION: Genes of pre-X interacting proteins in hepa-tocytes are successfully cloned and the results bring some new clues for studying the biological functions of pre-X and associated proteins.