【摘要】 实现产气肠杆菌(Enterobacter aerogenes)嘧啶核苷磷酸化酶(Pyri midine-nucleoside phosphoryl-ase,PyPNase)基因在大肠杆菌中的高效表达。用来源于产气肠杆菌的核苷磷酸化酶N末端序列与NCBI中公布的已知序列进行比对,设计一对引物,以该菌株基因组DNA为模板扩增出PyPNase基因。定向克隆到PET-28a(+)载体后转化到表达宿主大肠杆菌BL21中,筛选阳性克隆并测序,然后在LB培养基中优化发酵条件。结果:目的基因片断长度为1 302 bp。表达产物经SDS-PAGE和酶活测定表明PyPNase基因在大肠杆菌中获得活性表达,43 ku处有表达蛋白。37℃培养2 h至对数生长中期,降温到31℃,0.1 mmol/L IPTG诱导5h获得最大酶活力为512.3 U/mg,为出发菌株的17.2倍。重组PyPNase的成功表达为今后大规模生产5-FUR打下了基础。更多还原

【Abstract】 To obtain the recombinant PyPNase of Enterobacter aerogenes in large quantity in Escherichia coli,PyPNase gene from Enterobacter aerogenes was amplified by polymerase chain reaction(PCR) with the gene specific primer.The fragment was inserted into PET-28a(+) and then transferred into expressive strain Escherichia coli BL21.The fermentation conditions were optimized in LB medium.The expressed product was identifid by SDS-PAGE and enzyme activity was detected.The sequence encoding PyPNase of the initial strain is 1302bp.SDS-PAGE analysis and PyPNase activity assay showed that about 43ku protein was obtained.The maximal enzyme activity of 512.3U/mg could be obtained when keeping temperature at 37℃ for 2 hours in the growth phase and then shifting to 31℃ with 0.1mmol/L IPTG induced for 5 hours.Compared with the original Enterobacter aerogenes,the PyPNase activitiy from transformed E.coli was increased by 17.2 folds.The success of recombinant PyPNase has laid a foundation for the biosynthesis of 5-fluoro-uridine on a large scale.更多还原