摘要
目的:乙型肝炎病毒(HBV)核心蛋白(HBcAg)在HBV感染的肝细胞中可同时存在于筛选并克隆人肝细胞cDNA文库中与HBcAg相互作用蛋白的基因。方法:用多聚酶链反应(PCR)法扩增HBcAg基因,连接入酵母表达载体pGBKT7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X-α-gal)上进行双重筛选阳性菌落,PCR从中扩增出目的片段并测序,进行生物信息学分析。结果:成功克隆出HBcAg基因并在酵母细胞中表达,配合后选出既能在四缺(SD/-Trp-Leu-Ade-His)培养基又能在铺有X-α-gal的四缺培养基上均能生长,并变成蓝色的真阳性菌落16个,其中含金属硫蛋白A2基因的菌落有2个、未知基因4个、补体8α基因1个、NAD(P)H脱氢酶亚基1个、维甲酸受体应答元件2基因1个、细胞色素c氧化酶基因1个、细胞色素b基因1个、DAZ相关蛋白2基因2个、线粒体核蛋白L41基因2个、人血白蛋白基因1个。结论:成功克隆出乙型肝炎病毒核心蛋白的结合蛋白,为进一步研究HBcAg在病毒装配、损害肝细胞、感染致病等方面的具体作用提供了新线索。
AIM:TO screen proteins in hepatocytes interacting with hepatitis B virus core protein (HBcAg) with yeast-two hybrid technique for investigating the biological functions of HBcAg. METHODS:The HBcAg gene was amplified by polymerase chain reaction (PCR) and HBcAg bait plasmid was constructed with yeast-two hybrid system 3, then transformed into yeast AH109. The transformed yeast mated with yeast Y187 containing liver cDNA library plasmid in 2×YPDA 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:Sixteen colonies were sequenced, of which, two colonies were metallothionein 2A, one NAD(P)H dehydrogenase, one complement component 8 α polypeptide, one retinoic acid receptor responder (tazarotene induced), one cytochromeb, one cytochrome c oxidase subunit Ⅱ, one albumin, two DAZ associated protein 2, two mitochondrial ribosomal protein L41 and four new genes with unknown function. CONCLUSION:Genes of HBcAg interacting proteins in hepatocytes were successfully cloned and the results provided some new clues for studying the biological functions of HBcAg and its associated proteins.
出处
《世界华人消化杂志》
CAS
2003年第4期426-429,共4页
World Chinese Journal of Digestology
