糖皮质激素受体结合域诱饵表达载体的构建及鉴定  

Construction and identification of bait expression vector of glucocorticoid receptor binding domain

作　　者：郝坡[1] 刘北忠[1] 欧阳峰[2] 王东生[1] 刘畅[1] 钟梁[1] 金丹婷[1] 王翀 

机构地区：[1]重庆医科大学医学检验系临床生物化学教研室,临床检验诊断学省部共建教育部重点实验室,重庆400016 [2]广东省人民医院皮肤性病科实验室,广州510000

出　　处：《第三军医大学学报》2008年第3期216-219,共4页Journal of Third Military Medical University

基　　金：国家自然科学基金(30300449);国家中医药管理局基金(02-03ZP52)~~

摘　　要：目的构建糖皮质激素受体(glucocorticoid receptor,GR)结合域的诱饵表达载体。方法RT-PCR扩增GR结合域,克隆入pMD18-T,测序正确后,再亚克隆入诱饵载体pGBKT7中,再把构建好的诱饵载体pGBKT7-GR转化到酵母AH109细胞中,并用Western blot分析诱饵蛋白的表达情况,同时检测诱饵蛋白的毒性和自激活作用。结果成功扩增了GR结合域,并分别成功克隆到pMD18-T和pGBKT7中,测序结果正确。诱饵载体成功转化到酵母AH109细胞中,无毒性和自激活作用,Western blot分析结果也证实了酵母细胞高表达诱饵蛋白。结论成功构建了GR结合域的酵母诱饵表达载体。Objective To construct the bait expression plasmid pGBKTT-GR of glucocorticoid receptor （GR） binding domain. Methods The fragments of GR binding domain was amplified by RT-PCR, and then was cloned into pMD18-T. After being verified by sequencing, it was subcloned into the bait expression vector pGBKTT. Then the bait vector pGBKTT-GR was transformed into AH109 yeast cells and the expression of the bait protein was analyzed by Western blot. Toxicity and self-activation of the bait protein were detected. Resuits GR binding domain was amplified and cloned into pMD18-T and pGBKT7 successfully. The bait vector was transformed into AH109 yeast cells successfully, without toxicity or self-activation. The expression of the bait protein was confirmed by Western blot. Conclusion The successful construction of bait expression vector of glucocorticoid receptor binding domain lays the foundation for constructing small molecule ligand yeast threehybrid system.

关 键 词：糖皮质激素受体结合域 诱饵载体 诱饵蛋白 

分 类 号：R394-33[医药卫生—医学遗传学] R394.3[医药卫生—基础医学]