Tan H R, He S, Zhuang Z H, Xue Y G, Zhang Q J
Institute of Microbiology, Academia Sinica, Beijing.
Yi Chuan Xue Bao. 1990;17(5):390-7.
E. coli glucose isomerase gene was cloned into Streptomyces lividans using shuttle plasmid vector pSE-3. First plasmid pXI203 was constructed in E. coli using plasmids pXI200 (containing 1.6 kb glucose isomerase gene) and pGEM-3 digested with EcoR1. Then, recombinant plasmid pSEX100 was also constructed in E. coli using plasmids pXI203 and pSE-3 digested with HindIII. When the pSEX100 was transformed into Streptomyces lividans protoplasts, recombinants were obtained on R5YE medium containing 50 micrograms/ml neomycin and 50 micrograms/ml thiostrepton. The results showed that the E. coli glucose isomerase gene cloned and expressed in Streptomyces lividans via the analysis of restriction enzyme digestion as well as the detection of the glucose isomerase activity.
利用穿梭质粒载体pSE-3将大肠杆菌葡萄糖异构酶基因克隆到变铅青链霉菌中。首先，在大肠杆菌中使用经EcoR1消化的质粒pXI200（含1.6 kb葡萄糖异构酶基因）和pGEM-3构建质粒pXI203。然后，同样在大肠杆菌中使用经HindIII消化的质粒pXI203和pSE-3构建重组质粒pSEX100。当将pSEX100转化到变铅青链霉菌原生质体中时，在含有50微克/毫升新霉素和50微克/毫升硫链丝菌素的R5YE培养基上获得了重组体。通过限制性酶切分析以及葡萄糖异构酶活性检测结果表明，大肠杆菌葡萄糖异构酶基因已在变铅青链霉菌中克隆并表达。
