枯草芽孢杆菌葡萄糖脱氢酶基因的克隆及在大肠杆菌中的高效表达(英文)

CLONING AND HIGH LEVEL EXPRESSION OF GLUCOSE DEHYDROGENASE GENE FROM BACILLUS SUBTILIS IN E. COLI

利用PCR技术扩增得到来源于枯草芽孢杆菌的葡萄糖脱氢酶基因片段,并构建重组质粒pUC-T-GDH,然后将基因片段克隆于大肠杆菌表达载体pBV220中,得到表达质粒pBV-GDH.葡萄糖脱氢酶在含有表达质粒的基因工程菌株中得以诱导表达,聚丙烯酰胺凝胶电泳及薄层扫描结果表明,经诱导表达的葡萄糖脱氢酶约占基因工程菌株总蛋白的45％.葡萄糖脱氢酶活力测试表明,基因工程菌株无细胞抽提液中葡萄糖脱氢酶的活力为7.8U／毫克,约为对照组的30倍.实验结果初步说明,广泛应用于工业化生产的葡萄糖脱氢酶可以在基因工程菌株中得以大量表达并维持较高活力.

The gene of glucose dehydrogenase (GDH) from Bacillus subtilis was amplified by polymerase chain reaction (PCR) and cloned into the plasmid pUC-T then sub-cloned into the expression vector pBV220 to give a high level expression of the native protein in E. coli. The SDS-PAGE and thin layer scanning experiments showed that the amount of GDH was up to about 45% of the total proteins from transformants. The activity of GDH from cell-free extract of the genetic engineering Escherichia coli was around 7. 8U/mg, 30-fold higher than that of the control. The results showed that GDH, a widely used enzyme in industry, has been expressed efficiently in E. coli.