人Aurora B基因的克隆、原核表达及生物信息学分析  

Cloning,Prokaryotic Expression and Bioinformatics Analysis of Human Aurora B Gene

作　　者：邱贤秀[1,2] 周莹[1] 王雪琦[1] 丁伟超[1] 刘忠[1] 王绍祥[1] 王一飞[1] 

机构地区：[1]暨南大学生物医药研究开发基地,广东广州510632 [2]暨南大学药学院,广东广州510632

出　　处：《生物技术》2012年第5期5-10,共6页Biotechnology

基　　金：国家"十二.五"科技支撑计划项目("基于南药小分子化合物的抗单纯疱疹病毒药靶发现及药物开发";SQ2011SF12B02099);广东省自然科学基金青年基金项目("基于靶标TrxR的新型Mansonone F类似物抗宫颈癌作用机制研究";10451063201005506);国家自然科学基金项目("内质网应激在SNX-2112诱导食管癌细胞死亡中的作用";81201727)资助

摘　　要：目的:克隆出人Aurora B基因,并将其于大肠杆菌Rosetta(DE3)菌株中表达,获得重组蛋白。方法:利用TRIzol Reagent法提取食管癌ECa 109细胞总RNA,RT-PCR后以其cDNA为模板PCR扩增Aurora B基因,构建pET 28a(+)-Aurora B重组质粒,将其转化Rosetta(DE3)菌株,IPTG体外诱导表达重组蛋白。利用生物信息学工具对Aurora B的核酸序列和蛋白功能结构进行分析。结果:成功构建了pET 28a(+)-Aurora B重组质粒,经序列比对,与GenBank上公布的人Aurora B基因序列同源性达到了99%;获得Aurora B蛋白,大小约39kDa,蛋白表达量约占菌体总蛋白的28%。结论:对人Aurora B基因进行克隆、原核表达及生物信息学分析,为深入研究Aurora B在细胞周期调控中的作用机制及后续Aurora B抑制剂的体外筛选奠定基础。Objective： To clone human Aurora B gene, process prokaryotic expression in Rosetta （DE3 ）strain and get recombinant protein. Method：The total RNA was isolated by Trizol Reagent from ECa 109 cells,the cDNA was gained by RT - PCR and then used as a template for PCR,amplified Aurora B gene. Prokaryotic expression vector pET 28a（ ＋ ） -Aurora B was constructed and transformed into Rosetta（ DE3 ）. Induced by IPTG,the Aurora B protein was successfully expressed in vitro. Analyzed the nucleic acid sequence and proper- ties of Aurora B by bioinformatics analysis software. Result：The recombinant plasmid pET 28a（ ＋ ） - Aurora B was constructed,sequence alignment result showed that the sequence shares 99% of sequence homology with the Homo sapiens Aurora B reported in GenBank;Aurora B protein were obtained with the molecular weight which was about 39kDa, the expressed product contained about 28% of total somatic protein. Conclusion： The cloning, prokaryotic expression and bioinformatics analysis of Aurora B lay a foundation for the deeply study of its role in cell cycle regulation and the in vitro screening of Aurora B inhibitors.

关 键 词：极光激酶 B细胞周期 克隆 原核表达 生物信息学 

分 类 号：Q812[生物学—生物工程]