应用酵母双杂交技术筛选与前S1蛋白反式激活蛋白3结合的肝细胞蛋白基因

目的用酵母双杂交技术筛选白细胞中乙型肝炎病毒(HBV)前S1蛋白反式激活蛋白3(PS1TP3)的结合蛋白。方法通过聚合酶链反应扩增获得的PS1TP3基因,构建诱饵质粒pGBKT7-PS1TP3,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的质粒pACT2的单倍体酵母细胞Y187进行配合,在涂有X-α-Gal营养缺陷型培养基(SD/-Trp-Leu-His-Ade)上进行双重筛选,提取阳性酵母克隆的质粒转化大肠杆菌经BglII酶切后测序,进行生物信息学分析。结果筛选出30个与PS1TP3相互作用的蛋白,其中包括泛素蛋白酶E2、丝裂原活化蛋白激酶、β2-微球蛋白、磷酸酯酶张力蛋白等已知功能基因及4个未知功能序列,以及拼接新基因1个。结论成功克隆出PS1TP3蛋白的结合蛋白,为研究PS1TP3的分子生物学功能及HBV的致病机制提供了新的思路和线索。

Screening of gene encoding of hepatic proteins interacting with pre-S1 transactivated protein 3 via yeast two-hybrid

Objective To screen proteins in leukocytes interacting with hepatitis B virus(HBV) pre-S1 transactivated protein 3(PS1TP3)by yeast-two hybrid.Methods The DNA fragment of PS1TP3 was amplified by polymerase chain reaction.Bait plasmid pGBKT7-PS1TP3 was constructed and transform it to yeast cells AH109.Then mated it with yeast cells Y187 containing epatic cDNA library plasmid pACT2.The obtained diploid yeast cells were plated on synthetic dropout nutrient medium SD/-Trp-Leu-His-Ade containing X-α-Gal for selection twice.The plasmids of positive colonies were transformed to E.coli and digested by Bgl II enzyme,then sequenced and analyzed by bioinformatics.Results Thirty proteins binding to PS1TP3 were screened,including ubiquitin-conjugating enzyme E2,mitogen activated protein kinase,beta-2-microglobulin,phosphatase and tensin etc gene whose functions had been known and 4 unknown function series and one splice new gene.Conclusion Conjugated protein of PS1TP3 are successfully cloned and the results bring some new clues for studying the molecular biological functions of PS1TP3 and pathogenic mechanism of HBV.