产腈水合酶重组大肠杆菌的质粒稳定性研究  

Study on Plasmid Stability of a Recombinant Escherichia coli Producing Nitrile Hydratase

作　　者：史悦[1] 于慧敏[1] 田卓玲[1] 沈忠耀[1] 

机构地区：[1]清华大学化工系生物化工研究所,北京100084

出　　处：《中国生物工程杂志》2005年第8期70-75,共6页China Biotechnology

基　　金：国家自然科学基金青年基金资助项目(20206014);全国百篇优秀博士学位论文作者专项(200345)

摘　　要：成功构建了腈水合酶(nitrile hydratase,NHase)高表达的重组大肠杆菌E.coliBL21(DE3)/pETNHM(Kanr),研究了重组质粒pETNHM在重组菌株中的质粒稳定性。结果表明,pETNHM具有较好的结构稳定性,连续传代60代后质粒的基因序列没有明显缺失,且能够正常表达腈水合酶。pETNHM具有分离不稳定性,在无抗生素选择压力下,连续传代48代后质粒丢失的无质粒细胞开始出现。琼脂糖凝胶电泳定量分析表明,2/3的质粒pETNHM以二聚体形式存在,导致质粒拷贝数的下降。进一步研究表明,重组细胞的连续高速分裂及腈水合酶的高表达也会造成质粒拷贝数的下降,从而降低其分离稳定性。反之,重组菌株相对于宿主菌株的较高比生长速率有利于保持含质粒细胞的生长优势,卡那霉素的选择压力则能够保证质粒的稳定遗传。A novel recombinant E. coli BL21 （ DE3 ） / pETNH^M （ Kan^r） was successfully constructed for high expression of nitrile hydratase （NHase）, and the plasmid stability of pETNH^M was mainly focused on. Plasmid DNAs of pETNH^M after 60 generations didn＇t take on obvious gene deletion, indicating that plasmid pETNH^M is structurally stable. However, plasmid pETNH^M is segregationally unstable, because the plasmid-free cells appeared after 48 generations under no kanamycin selection pressure. Agarose gel electrophoresis showed that 2/3 of plasmids formed dimer form in the recombinant cells, which significantly cut down the plasmid copy number of pETNH^M. Further studies showed that the continuous high-speed division of recombinant cells and the high level expression of NHase would both reduce the copy number of plasmids and worsen the segregational instability of pETNH^M. On the contrary, the higher specific growth rate of recombinant cells was beneficial to keep the growing superiority of plasmid-harboring cells, and the supplementation of kanamycin pressure will ensure the stable inheritance of pETNH^M in the recombinant E. coli BL21 （DE3） / pETNH^M.

关 键 词：腈水合酶 重组大肠杆茵 质粒稳定性 分离不稳定性 质粒拷贝数 重组大肠杆菌 稳定性研究 大肠杆菌E.coli 琼脂糖凝胶电泳 重组菌株 

分 类 号：Q78[生物学—分子生物学] TQ464.7[化学工程—制药化工]