抗辐射菌属-大肠杆菌间的穿梭载体的构建及荧光基因的表达

Construction of deinococcal bacteria - Escherchia coli shuttle vector and expression of Luciferase gene

摘要:

摘要：【目的】构建抗辐射菌属一大肠杆菌间的穿梭载体，通过此载体使荧光素酶基因在大肠杆菌中得到表达。【方法】以质粒pUE30、pGBM5及pKatCAT为基础，构建抗辐射菌属一大肠杆菌间的穿梭载体，将groEL启动子和荧光素酶基因lux+插入到构建的穿梭载体中得到穿梭表达载体，并将该载体转化大肠杆菌诱导荧光素酶基因的表达。【结果】成功构建了大小约为5.8 kb的抗辐射菌属一大肠杆菌间的穿梭载体pZT17，该载体在没有抗生素的非选择性培养基中能稳定存在。在穿梭载体pZT17的EcoRV部位插入含有groEL启动子和荧光素酶基因lux+的DNA片段，构建得到了穿梭表达载体pZTGL2；利用该表达载体在大肠杆菌中可诱导表达荧光素酶基因。【结论】构建的穿梭表达载体为以后用大肠杆菌高效表达来源于抗辐射菌的基因、特别是DNA损伤修复蛋白基因，提供了可能。

Abstract:

Abstract: [0bjective] To express Luciferase gene in Escherchia coli through developed Deinococcal bacteria - E. coli shuttle expression vector. [Methods]The D. bacteria - E. coli shuttle expression vector pZT17 was constructed based on plasmids of pUE30, pGBM5 and pKatCAT. Then pZT17 with lux+ from Photinus pyralis was used to transform into D. grandis and E.coli. The recombinant strains were induced separately. [Results] Based on a small cryptic plasmid from Deinococcus radiopugnans, a shuttle vector between Escherichia coli and deinococcal bacteria was constructed. The plasmid vector could stably aintained in Deinococcus grandis under non-selective conditions. Moreover, it is showed that a luciferase gene was highly expressed both observed in D. grandis and E.coli. [Conclusions]The D. bacteria - E. coli shuttle vector was constructed successfully, the developed shuttle vector makes it possible to induce expression of DNA damage and repair gene from Deinococcus species.