【摘要】 目的 :运用分子生物学手段获得葡萄糖脱氢酶基因 ,并表达该基因。方法 :根据枯草芽孢杆菌葡萄糖脱氢酶基因序列设计引物 ,通过PCR获得该基因并与源序列进行比较分析 ,与表达载体pET2 2b连接后转化至大肠杆菌JM 10 9(DE3)进行表达 ,并在全自动生化分析仪上用速率法测定其活性。结果 :克隆到的葡萄糖脱氢酶基因与源基因的同源性为 99% ,克隆到的GDH编码的氨基酸序列 16 7位左右存在突变 :EVI(GAAGTGATT )→AF(GCGTTT) ,利用酶的初提液测定的葡萄糖脱氢酶活力为 75U/L ,比活性为 10U/mg。 结论 :运用基因工程手段获得了葡萄糖脱氢酶基因 ,与表达载体连接后的重组体经大肠杆菌初步表达有活力 ,经诱导有望获得高产量的葡萄糖脱氢酶 ,从而为临床服务。更多还原

【Abstract】 Objective:To obtain a gene encoding glucose dehydrogenase from genomic DNA of Bacillus subtilis and express it. Methods: Primers were designed based on the gene sequence and the gene of GDH was obtained using PCR technique, the pET22b vector combined with the GDH gene was transformed into JM109(DE3) to express. Results: The cloned gene has 99% homogenous with that from B.subtilis.The amino acid sequence according to gene sequence have mutagenesis in 167 EVI(GAAGTGATT)→AF(GCGTTT). The activity of primal enzyme was 75 U/L and the specific activity was about 10 U/mg. Conclusion: Based on PCR techniques, a gene encoding glucose dehydrogenase was obtained from genomic DNA of B.subtilis. The resulting recombinant E.coli gave a certain expression of GDH. By inducing, a high level expression of GDH was expected to reach.更多还原