应用Red重组技术构建四环素抗性多基因串联表达载体  

Construction of a tetracycline resistance multi-gene coexpression vector using Red recombineering

作　　者：何彰华[1] 师明磊[1] 王洋[1] 叶丙雨[1] 黄芬[1] 张彦[1] 范秋声[1] 王东[1] 肖丽霞[1] 张乐之[1] 李德彬[1] 闫兴[1] 赵志虎[1] 

机构地区：[1]军事医学科学院生物工程研究所,北京100071

出　　处：《军事医学》2011年第10期754-757,799,共5页Military Medical Sciences

基　　金：国家重大新药创制关键技术平台(2008ZXJ09007-01)

摘　　要：目的构建一种四环素抗性的多基因串联表达载体。方法设计同源臂引物,通过重叠延伸PCR获取含四环素抗性基因的打靶序列,应用pKD46介导的Red重组系统,在DH5α内替换多基因串联表达载体pET-m28a(+)中的抗性基因序列;再利用同尾酶(isocaudarner)策略,将sfp和accA1两基因串联组合于pET-mt28a(+)中,并进行共表达鉴定。结果获得了含四环素抗性的重组质粒pET-mt28a(+);构建了两基因串联重组质粒pET/sfp/accA1-mt28a(+),且两基因能够在同一载体上协同表达。结论成功构建了一种四环素抗性表达载体pET-mt28a(+),并可介导多基因的协同表达,为红霉素合成通路在大肠杆菌中的重建奠定了基础。ObjectiveTo construct a tetracycline resistance multi-gene coexpression vector.MethodsTetracycline resistance gene with homologous primers was cloned by overlap extension PCR before the resistance gene sequences in multi-gene coexpression vector pET-m28a（＋） were replaced by tetracycline resistance gene in DH5α via Red-mediated recombination system pKD46.Positive recombinants were selected under tetracycline pressure.Using isocaudarners,sfp and accA1 were combined in pET-mt28a（＋）,and the expression of genes was identified.ResultsPositive recombinants with pET-m28a（＋） and tetracycline resistance were selected.Restructuring plasmid pET/sfp/accA1-mt28a（＋）was constructed,and the two genes could co-express in one vector.ConclusionWe successfully construct a tetracycline resistance multi-gene coexpression vector pET-mt28a（＋）,contributing to the reconstruction of erythromycin synthesis pathways in E.coli.

关 键 词：四环素抗药性 多基因串联表达载体 RED重组 质粒 红霉素 

分 类 号：R915[医药卫生—微生物与生化药学]