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目的筛选并克隆人肝细胞cDNA文库中与乙型肝炎e抗原反式激活蛋白(HBeAgTP)相互作用蛋白的基因。方法应用抑制性消减杂交技术及生物信息学技术筛选并克隆HBeAg反式激活的新型靶基因HBeAgTP。用聚合酶链反应(PCR)法扩增HBeAgTP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HBeAgTP基因并在酵母细胞中表达,应用酵母双杂交筛选出阳性菌落24个,经生物信息学分析为15种已知基因。结论成功克隆出HBeAgTP的结合蛋白,包括一些与细胞内蛋白质的转录、翻译、免疫调节及物质和能量代谢相关的基因。
Objective To screen and clone the gene of protein interacting with hepatitis B e antigen transactivator protein (HBeAgTP) in human hepatocyte cDNA library. Methods Suppression subtractive hybridization and bioinformatics techniques were used to screen and clone a novel HBeAg transactivated target gene, HBeAgTP. The HBeAgTP gene was amplified by polymerase chain reaction (PCR), inserted into the yeast expression vector pGBKT7 to construct the bait plasmid, transformed and expressed in the yeast AH109, and then transformed into the yeast cell Y187 transformed with the human liver cDNA library plasmid pACT2 The positive colonies were double-screened on auxotrophic medium and X-α-galactose (X-α-gal). The target fragment was amplified by PCR and sequenced for bioinformatics analysis. Results The HBeAgTP gene was successfully cloned and expressed in yeast cells. Twenty-four positive colonies were screened by yeast two-hybrid system and 15 known genes were analyzed by bioinformatics analysis. Conclusion HBeAgTP binding protein was cloned successfully, including some genes related to intracellular transcription, translation, immunomodulation and substance and energy metabolism.