摘要
本文在大肠杆菌/枮草芽孢杆菌间的穿梭质粒pTG 402的基础上构建了几个新的带有显色标志基闲xylE的表达质粒,摸索了该基因所编码的邻苯二酚2,3-双加氧酶(CatO_2ase)的表达条件,分析了该酶一级结构与二级结构的亲水性和疏水性,测定了它在大肠杆菌中的产量与分布。结果表明,CatO_2ase与各质粒的表达量不等,表达量高低与培养时间、宿主菌及诱导与否等影响因素有关;表达后有部分酶可在胞外测出,但大部分仍定域于膜内,亲、疏水性分析示该酶不具分泌性蛋白的显著特点。因该酶易于检测和定量,可作为一种选择性标记和监测指示系统在基因工程中推广应用,同时亦为用基因工程菌消除芳烃类化合物的污染提供了理论依据。
A series of new plasmids containing xy/E gene was constructed based on the shuttle plas-mid pTG 402 between E. coli and B. subtilis. The expressed xylE gene product catechol 2,3-dio-xygenase (CatO2ase) was measured for its output and distribution, and analysed for its struc-trural hydrophobicity and hydrophilicity. It was demonstrated that the output of CatO2ase was relative to plasmids, host cells, culture time and with or without induction. The enzyme didn't have features of excretional proteins and was mostly distributed inside the cells though a little could be detected in culture medium. It can be used as a selective marker, indicator and monitor in the study of genetic engineering. This research also provides a scientific basis for eliminating pollution of aromatic hydrocabonic compounds with genetic engineering bacteria.
关键词
大肠杆菌
表达
邻菌二酶
双加氧酶
xylE gene, Catechol 2,3-dioxygenase, Expression, Distribution
