人类RhoxF1蛋白的生物信息学分析及其自激活检测  

Construction and Activity Test of Bait Plasmid of RhoxF1 Gene in Yeast Two-hybrid System

作　　者：苏宪礼[1] 郭芬[2] 刘兆宇[1] 陈孟璋[1] 张欣[1] 周天鸿[1] 李月琴[1] 

机构地区：[1]暨南大学基因工程药物国家工程研究中心,广东广州510632 [2]广东药学院生命科学与生物制药学院,广东广州510006

出　　处：《生物技术》2010年第3期39-42,共4页Biotechnology

基　　金：广东省自然科学基金项目(8151063201000013);"211"工程经费项目资助

摘　　要：目的:生物信息学分析人类RhoxF1蛋白的氨基酸序列,构建RhoxF1酵母双杂交诱饵载体,并检测其自激活活性。方法:生物信息学分析RhoxF1蛋白保守区域,比较小鼠Rhox5与人RhoxF1的氨基酸序列同源性,PCR扩增RhoxF1基因,与PMD-18-T载体连接,然后把RhoxF1克隆到pGBKT7载体中,醋酸锂法将重组质粒pGBKT7-RhoxF1转入酵母菌AH109,观察pG-BKT7-RhoxF1在AH109中的表达情况,检测诱饵载体有无毒性和自激活作用。结果:RhoxF1蛋白保守区域有大量的DNA结合位点,与Rhox5具有30%的同源性。成功构建诱饵载体pGBKT7-RhoxF1,目的片段可在酵母AH109中表达,并且对酵母菌无毒性,对报告基因无自激活功能。结论:探索了RhoxF1的蛋白特性,成功构建pGBKT7-RhoxF1重组质粒,可以作为酵母双杂交诱饵载体筛选与RhoxF1相互作用的蛋白,进而对其进行功能性研究。Objective：To analyze the sequences of RhoxF1 protein,construct the bait plasmid of RhoxF1 in yeast two-hybrid system and test its self-activation acitivity.Method：Conserved domains were analyzed and the homology between mouse Rhox5 protein and human RhoxF1 protein was compared by bioinformatics method.PCR was used to amplify RhoxF1 gene and the fragment was inserted into the PMD 18-T vector.After verified,the fragment was subcloned into pGBKT7 vector.After that,the 1 plasmid was transformed into the yeast strain Saccharomyces cerevisiae AH109 and its toxicity and transcriptional activation was tested by both the phenotype assay and β galactosidase assays.Result：Several conserved DNA binding sites were found and the homology between Rhox5 and RhoxF1 amounted up to 30%.The bait plasmid pGBKT7-RhoxF1 was constructed correctly.Activity test of β-galactosidase revealed that pGBKT7-RhoxF1 couldn＇t be self-activated.Conclusion：Human RhoxF1 protein has the high homogeneous characteristics with mouse Rhox5 protein.In addition,pGBKT7-RhoxF1,as a bait plasmid of the yeast two-hybrid system,can be used to screen the protein interacting with RhoxF1.

关 键 词：基因 RhoxF1 酵母双杂交 生物信息学 自激活 

分 类 号：Q71[生物学—分子生物学]