| 应用Red重组技术构建四环素抗性多基因串联表达载体 |
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| 引用本文： | 何彰华,师明磊,王洋,叶丙雨,黄芬,张彦,范秋声,王东,肖丽霞,张乐之,李德彬,闫兴,赵志虎.应用Red重组技术构建四环素抗性多基因串联表达载体[J].军事医学科学院院刊,2011,35(10):754-757,799. |
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| 作者姓名： | 何彰华 师明磊 王洋 叶丙雨 黄芬 张彦 范秋声 王东 肖丽霞 张乐之 李德彬 闫兴 赵志虎 |
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| 作者单位： | 军事医学科学院生物工程研究所,北京,100071 |
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| 基金项目： | 国家重大新药创制关键技术平台(2008ZXJ09007-01) |
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| 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。
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| 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 |
Construction of a tetracycline resistance multi-gene coexpression vector using Red recombineering |
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| HE Zhang-hua,SHI Ming-lei,WANG Yang,YE Bing-yu,HUANG Fen,ZHANG Yan,FAN Qiu-sheng,WANG Dong,XIAO Li-xia,ZHANG Le-zhi,LI De-bin,YAN Xing,ZHAO Zhi-hu.Construction of a tetracycline resistance multi-gene coexpression vector using Red recombineering[J].Bulletin of the Academy of Military Medical Sciences,2011,35(10):754-757,799. |
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| Authors: | HE Zhang-hua SHI Ming-lei WANG Yang YE Bing-yu HUANG Fen ZHANG Yan FAN Qiu-sheng WANG Dong XIAO Li-xia ZHANG Le-zhi LI De-bin YAN Xing ZHAO Zhi-hu |
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| Institution: | HE Zhang-hua,SHI Ming-lei,WANG Yang,YE Bing-yu,HUANG Fen,ZHANG Yan,FAN Qiu-sheng,WANG Dong,XIAO Li-xia,ZHANG Le-zhi,LI De-bin,YAN Xing,ZHAO Zhi-hu* (Beijing Institute of Biotechnology,Beijing 100071,China) |
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| Abstract: | 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. |
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| Keywords: | tetracycline resistance multi-gene coexpression vector Red recombineering plasmids erythromycin |
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