乙型肝炎病毒前S2蛋白结合蛋白基因的筛选

Screening of the genes of hepatitis B virus PreS2 interacting proteins

目的筛选并克隆人肝细胞中与乙型肝炎病毒前S2蛋白(HBV PreS2)相互作用的蛋白质基因。 方法用聚合酶链反应(PCR)技术扩增HBV PreS2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cDNA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,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HBVPreS2基因并在酵母细胞中表达,配合后选出既能在4缺(SD/-Trp-Leu-Ade-His)培养基又能在铺有X-α-半乳糖(X-α-gal)的4缺培养基上生长并变成蓝色的真阳性菌落26个,其中含金属硫蛋白A2基因的菌落有12个,细胞色素C氧化酶Ⅱ有1个,细胞色素:P450 IVF亚基2个,细胞色素C氧化酶Ⅳ亚基同功酶1有2个,人血白蛋白3个,。Na+-K+ATP酶β1亚基1个,前清蛋白2个,植物血凝素半乳糖苷结合亚基1个,未知基因2个。结论 成功克隆出HBV PreS2蛋白的结合蛋白,为进一步研究HBV PreS2的作用提供了新线索。

Objective To screen and clone the genes of proteins in hepatocytes interacting with hepatitis B virus (HBV) PreS2 by yeast-two hybridizition technique. Methods The HBV PreS2 gene was amplified by polymerase chain reaction (PCR) and HBV PreS2 bait plasmid was constructed by using yeast-two hybridization system 3, then transformed into yeast AH109, followed by mating with yeast Y187 containing liver cDNA library plasmid in 2 x YPDA medium. Dip-loid yeast was plated on synthetic dropout nutrient medium (SD/-Trp-Leu-Ade-His) and synthetic dropout nutrient medium (SD/-Trp-Leu-Ade-His) containing X-a-gal for selecting positive blue clones, then amplified by PCR, sequenced, and performed bioinformatics analysis. Results HBV PreS2 gene was cloned successfully and expressed in yeast AH109. Tweenty-six positive colonies were selected, among them, twelve containing metallothionein 2A, one cytochrome C oxidase n , two cytochrome P450 subfamily IVF, two cytochrome c oxidase subunit IV isoform 1, three albumin (A, one Na+K+ transporting ATPase beta-1 polypeptide, two prealbumin, one lectin galactoside-binding subunit, and Two new genes with unknown function. Conclusion Genes of HBV PreS2 interacting proteins have been successfully cloned, which brings some new clues for studying the biological functions of HBV PreS2 and related proteins.