| 苏春,余佳,邱荣国,唐莉.Red/ET同源重组系统介导的质粒载体快速构建[J].,2013,53(1): |
| Red/ET同源重组系统介导的质粒载体快速构建 |
| Rapid construction of plasmid vector by Red/ET mediated in vivo homologous recombination |
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| DOI：10.7511/dllgxb201301003 |
| 中文关键词: Red/ET技术 同源重组 抗性基因 一步融合 正反选择标记 克隆 |
| 英文关键词: Red/ET technology homologous recombination resistance gene one-step gene fusion positive-negative selectable marker cloning |
| 基金项目:国家杰出青年科学基金资助项目(30688003). |
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| 中文摘要: |
| Red/ET重组技术是近年来建立的一种基于高效率体内同源重组的新型遗传工程技术,该技术简便、快速．首先,通过一步融合的方法将链霉素抗性基因和氯霉素抗性基因同时克隆至pUC19上,从而构建出含有正反选择标记的载体pRC；其次,用安普霉素抗性基因或者氯霉素抗性基因替换了常用表达载体pET-28b上的卡那霉素抗性基因,改变了其抗性选择标记,得到的表达载体pMT和pCT可以更广泛地应用于不同宿主中；最后,还将启动和终止表达的区域定点插入pACYC184载体中,使其成为可以独立表达蛋白的表达载体p184．本研究所得到的载体为大肠杆菌宿主菌中的基因克隆和蛋白表达提供了基础． |
| 英文摘要: |
| Red/ET recombination technology developed in recent years is a novel genetic engineering technique, which facilitates rapid and easy genetic manipulation based on efficient in vivo homologous recombination. Streptomycin resistance gene and chloramphenicol resistance gene are cloned to pUC19 by one-step gene fusion to construct a new vector with both positive and negative selectable markers, which is designated as pRC. Besides, kanamycin resistance gene of pET-28b is replaced by apramycin or chloramphenicol resistance gene to create pMT and pCT for more flexible screening in different hosts. Finally, expression vector pACYC184 is modified by inserting a promoter and a terminator sequence, respectively. The vectors constructed provide basis for versatile gene clone and protein expression in different host E. coli strains. |
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