利用酵母双杂交技术筛选与转录因子HOXA11相互作用蛋白  被引量:2

Screening of proteins interacting with HOXA11 by yeast two-hybrid system

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作  者:程志[1] 高蕊[2] 马林强[2] 黄慧哲[2] 

机构地区:[1]重庆医科大学基础医学院细胞生物学与遗传学教研室,重庆400016 [2]重庆医科大学发育生物学研究室,重庆400016

出  处:《重庆医科大学学报》2016年第5期443-447,共5页Journal of Chongqing Medical University

基  金:国家自然科学基金资助项目(编号:81300463);重庆市科委自然科学基金资助项目(编号:cstc2013jcyj A10086);重庆市杰出青年科学基金资助项目(编号:cstc2012jjjq10001)

摘  要:目的:利用酵母双杂交技术从均一化的人肾透明细胞腺癌c DNA文库中筛选与转录因子HOXA11相互作用的蛋白。方法:培养人肾透明细胞腺癌细胞,提取RNA,采用SMART技术制备均一化的人肾透明细胞腺癌c DNA文库。同时,聚合酶链反应扩增人HOXA11基因片段(1~846 bp),将此基因片段重组入p GBKT7载体,构建诱饵质粒p GBKT7-HOXA11,经酶切和测序验证质粒构建正确。将诱饵质粒转化酵母菌AH109,检测其有无毒性与自激活作用。随后,利用酵母双杂交系统从c DNA文库中筛选与HOXA11相互作用的蛋白。对筛选到的阳性克隆进行回转验证以及测序分析。结果:成功构建了人肾透明细胞腺癌c DNA文库和p GBKT7-HOXA11诱饵质粒。在AH109酵母细胞中,该质粒无毒性、无自激活作用。酵母双杂交筛选获得了5个与HOXA11相互作用阳性克隆。结论:获得人肾透明细胞腺癌细胞中与HOXA11相互作用蛋白,为进一步研究HOXA11在个体发育以及癌症发生发展中的作用机制奠定了基础。Objective:To screen proteins binding with HOXA11 from human renal clear cell adenocarcinoma c DNA library by yeast two-hybrid system. Methods:The RNA of human renal clear cell adenocarcinoma cell was obtained and then transformed into c DNA library using SMART technology. Simultaneously,the c DNA fragments of HOXA11(1-846 bp)were amplified by polymerase chain reaction and constructed into p GBKT7 vector as the bait plasmid. The bait plasmid was transferred into yeast AH109 strain. Its toxicity and autoactivation were tested. Subsequently,the c DNA library was screened with p GBKT7-HOXA11 as bait plasmid by yeast twohybrid system. Finally,the positive clones were sequenced and further analyzed. Results:Human renal clear cell adenocarcinoma c DNA library and bait plasmid p GBKT7-HOXA11 were constructed successfully. The bait plasmid has no toxicity or autoactivation in AH109.Five positive clones were obtained and validated. Conclusion:Proteins deduced to interact with HOXA11 are identified through yeast two-hybrid screening. These results help to elucidate the possible roles of HOXA11 in carcinogenesis and development.

关 键 词:HOXA11 SMART技术 酵母双杂交 

分 类 号:R737.1[医药卫生—肿瘤]

 

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