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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 and express it in E. coli Rosetta (DE3) strain to obtain recombinant protein. Methods: Total RNA was extracted from esophageal cancer cells (ECa 109) by TRIzol Reagent method. The cDNA of Aurora B was amplified by RT - PCR. The recombinant plasmid pET28a (+) - Aurora B was constructed and transformed into Rosetta (DE3) Strain, IPTG induces the expression of recombinant protein in vitro. The bioinformatics tools were used to analyze the nucleotide sequence and protein structure of Aurora B. Results: The recombinant plasmid pET 28a (+) - Aurora B was successfully constructed. The sequence of human Aurora B gene was 99% homologous to that published in GenBank. Aurora B protein was obtained with a size of about 39 kDa and protein expression About 28% of total bacterial protein. CONCLUSION: Cloning, prokaryotic expression and bioinformatics analysis of human Aurora B gene lay the foundation for further studies on the mechanism of Aurora B in cell cycle regulation and screening of Aurora B inhibitors in vitro.