假肠膜明串珠菌甘露醇脱氢酶基因的克隆及表达

摘要

The structural gene encoding mannitol dehydrogenase (MDH) from Leuconostoc pseudomesenteroides was amplified by PCR and cloned into the vector pETDuet-1. As a result, the plasmid pETDuet-1-mdh was constructed and transformed into E. coli BL21(DE3) for MDH expression induced by isopropyl-β-D-thiogalactoside (IPTG). The length of the mdh structural gene was l 017 bp. The recombinant mdh gene was successfully expressed in Escherichia coli and the molecular weight of the expressed protein was 36.0 kD. The activity of recombinant mannitol dehydrogenase was 0.15 U/mg pro, which was higher than the MDH activity of Leuconostoc pseudomesenteroides (0.03 U/mg pro).%利用聚合酶链式反应从假肠膜明串珠菌中扩增出甘露醇脱氢酶（mannitol dehydrogenase，MDA）基因的结构基因，克隆入表达载体pETDuet-1，构建了甘露醇脱氢酶表达质粒pETDuet-1-mdh，将其转化进入大肠杆菌BL21(DE3)，经异丙基-β-D-硫代半乳糖苷诱导表达。假肠膜明串珠菌甘露醇脱氢酶结构基因长度为1017 bp，重组甘露醇脱氢酶基因在大肠杆菌内成功表达，其蛋白质分子质量为36.0 kD；重组甘露醇脱氢酶活力为0.15 U/mg pro，高于假肠膜明串珠菌中甘露醇脱氢酶活力0.03 U/mg pro。