丙型肝炎病毒E2结合蛋白1结合蛋白的酵母双杂交筛选研究

Screening and cloning of the genes of protein interacting with the envelope 2 binding protein 1 of hepatitis C virus

目的 应用酵母技术筛选丙型肝炎病毒 (HCV) 7包膜蛋白E2结合蛋白 1(E2BP1)的结合蛋白。方法 应用酵母双杂交系统 3 ,构建E2BP1诱饵质粒 ,转化酵母AH10 9,与含人肝细胞cDNA文库质粒的酵母Y187进行配合 ,于涂有x α gal营养缺陷型培养基 (SD/ Trp Leu His Ade)上筛选生长 ,对于阳性克隆的插入基因片段序列进行测定 ,并进行生物信息学分析。结果 5 3个阳性克隆测序 ,结果与GenBank数据库进行初步比较。其中 6个克隆为未知功能基因 ,其余 47个均与己知基因的序列高度同源 ( 90 %～10 0 % )。这 47个己知基因包括人类酶原颗粒蛋白 16、人类染色体序列、人类乙酰辅酶A转乙酰酶 1(ACAT1)、人类 4 羟基苯丙酮酸二加氧酶、人类硒蛋白P1(SEPP1)、人类组织蛋白酶F(CTSF)、人类prosaposin(PSAP)、人类金属硫蛋白 2A等 16种。

Objective To screen and clone the binding protein of hepatitis C virus (HCV) envelope protein 2-binding protein 1 (E2BP1) by using yeast two-hybrid techniques.Methods To investigate the biological function of E2BP1, we performed yeast two hybrid to seek for proteins in hepatocytes interacting with E2BP1. We constructed E2BP1 bait plasmid by cloning the gene of E2BP1 into pGBKT7, and transformed it into yeast AH109(a type). The transformed yeast mated with yeast Y187(a type)containing liver cDNA library plasmid in 2×YPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing x-a-gal for selection and screening.Results Fifty-three colonies were sequenced,among which, six colonies were new genes with unknown function.Conclusion These results will pave the way to study the molecular mechanism of E2BP1 protein of HCV and to develop new therapy for chronic hepatitis C.