hERR1/HBD酵母表达质粒的构建及初步鉴定  

Construction of yeast expression plasmid hERR1/HBD and preliminary identification of its function

作　　者：陈敏[1] 李渝萍[1] 陈健[1] 陈彬[1] 周度金[1] 

机构地区：[1]第三军医大学基础医学部生物化学与分子生物学教研室,重庆400038

出　　处：《第三军医大学学报》2003年第1期51-53,共3页Journal of Third Military Medical University

基　　金：国家自然科学基金资助项目 ( 30 0 0 70 59)

摘　　要：目的　构建酵母双杂合系统诱饵蛋白表达质粒pGBT9 hERR1 HBD。方法　PCR扩增hERR1 HBD(激素激活域 )cDNA ,与酵母Gal4 DBD(DNA结合域 )表达质粒pGBT9重组构建融合蛋白表达质粒 ,酶切、测序鉴定后 ,用于酵母双杂合分析 ,检查其自主转录激活作用及与已知hERR1辅活化子PNRC的相互作用。结果　该质粒有正确的阅读框 ,其表达蛋白不具有自主转录激活作用 ,与已知核受体辅活化子PNRC有明显的相互作用。结论　成功构建了表达质粒pGBT9 hERR1 HBD ,并证实该质粒可作为酵母双杂合系统的诱饵蛋白质粒 ,用于乳腺组织中与hERR1有相互作用的蛋白质基因的筛选。Objective To construct the expression plasmid pGBT9 hERR1/HBD as a bait in yeast two hybrid system. Methods A cDNA fragment encoding for hERR1/HBD was amplified by PCR and inserted into Gal4/DBD of yeast expression plasmid pGBT9. After identification of the positive clone by digestion of restriction endonuclease and sequencing, its transcriptional activation and the interaction with a known coactivator PNRC were tested in yeast two hybrid assays. Results The protein expressed by the plasmid which had a correct reading frame, had no transcriptional activation and could interact with a known coactivator PNRC. Conclusion Plasmid pGBT9 hERR1/HBD, which encodes fusion protein of Gal4DBD and hERR1/HBD, is successfully constructed. This fusion protein is demonstrated to be suitable as a bait in yeast two hybrid screening of the protein interacting with ERR1 from breast tissue library.

关 键 词：酵母表达质粒 乳腺癌 雌激素受体相关受体-1 酵母双杂合系统 乳腺肿瘤 聚合酶链反应 

分 类 号：R737.9[医药卫生—肿瘤]