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

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.