【摘要】 目的为获得较高活力的葡萄糖脱氢酶。方法根据枯草芽孢杆菌葡萄糖脱氢酶基因两端序列设计引物,以枯草芽孢杆菌WB600基因组DNA为模板,通过聚合酶链反应(PCR)获得该基因,与表达载体pET22b连接后转化至大肠杆菌JM109(DE3)进行表达,并在半自动生化分析仪上用速率法测定其活性。结果聚丙烯酰胺凝胶电泳(SDS-PAGE)显示,重组菌能表达单体分子量为31.5kD的特异性蛋白。通过对重组基因表达条件的研究发现,诱导剂(异丙基硫代-β-D-半乳糖苷)浓度为0.5mmol/L,诱导2.5~3h后用240W超声破碎细胞,可实现该基因的较高表达。结论运用基因工程手段获得了葡萄糖脱氢酶基因,重组基因经大肠杆菌诱导表达,获得了较高活力的葡萄糖脱氢酶,经纯化后可为临床服务。更多还原

【Abstract】 Objective To obtain the glucose dehydrogenase(GDH) with higher activity.Methods To gain the gene encoding glucose dehydrogenase from genomic DNA of Bacillus subtilis WB600 by PCR.To ligate with vector pET22b and transducted into E.coli JM109 to express it.Results SDS-PAGE showed the subunit molecular weight of GDH was 31.5 kDal. When IPTG concentration was 0.5 mmol/L and induction time was 2.5-3.0 h,it was the more optimal expression condition.Conclusion Based on PCR techniques, a gene encoding glucose dehydrogenase was obtained. By inducing, a high level expression of GDH was reached.更多还原