遗传 ›› 2006, Vol. 28 ›› Issue (1): 71-77.

• 研究报告 • 上一篇    下一篇

pBR322-Red介导的E.Coli染色体基因敲入、位点及表达研究

陈 伟1;2;李山虎1;于 梅1;王鸣刚2;周建光1   

  1. (1. 军事医学科学院生物工程研究所,北京 100850;2. 厦门大学生命科学学院细胞生物学与肿瘤细胞工程教育部重点实验室, 厦门361005)
  • 收稿日期:2004-11-02 修回日期:2005-01-16 出版日期:2006-01-10 发布日期:2006-01-10
  • 通讯作者: 陈伟

pBR322-Red Mediated Gene Knockin,Sites And Expression in E.coli Chromosome

CHEN Wei1, 2, LI Shan-Hu1, YU Mei1, WANG Ming-Gang2, ZHOU Jian-Guang1   

  1. (1. Institute of Biotechnology, Academy of Military Medical Sciences, Beijing 100850,China; 2.The key laboratory of Education Ministry for Cell Biology and Tumor Cell Engineering, School of Life Sciences, Xiamen Univ. Xiamen 361005,China)
  • Received:2004-11-02 Revised:2005-01-16 Online:2006-01-10 Published:2006-01-10

摘要: 应用pBR322-Red介导的重组工程系统,Kan/sacB选择反选择系统,双链线性DNA重组技术和重叠引物介导的DNA重组技术,将长度为1 653 bp的luc报告基因分别敲入到E.coli W3110染色体lacZ, lacY和lacA基因的位置,建立了一系列具有新遗传表型的菌株:CWL2、CWL4和CWL6。荧光素酶分析表明,外源报告基因luc能在这3个结构基因处有效的组成型表达。为了进一步确定外源基因的表达情况,用霍乱毒素B亚单位基因ctxb替换了lacZ基因,构建了新菌株CWD1。证明了以单拷贝形式存在在大肠杆菌染色体CWD1上的ctxb基因能有效的表达CTB蛋白并能将其分泌至细胞外培养液中。结果初步确定了大肠杆菌染色体上的lac操纵子结构基因位点适合外源基因的敲入和表达。

关键词: 霍乱毒素B亚单位, 基因敲入, 重组工程, pBR322-Red

Abstract: Genes lacZ,lacY and lacA in the lac opron of E.coli chromosome were respectively substituted with gene luc by using plasmid pBR322-Red, selection-counterselection system kan/sacB and various strategies of Red homologous recombination including Red mediated linearized double-stranded DNA homologous recombination and Red mediated recombineering with overlapping single stranded DNA oligonucleotides.Then, a series of new strains, CWL2,CWL4 and CWL6, were constructed and we found that they can express protein Luc efficiently. To further study the expression of exogenous genes at the site of lacZ, we have constructed a strain named CWD1 by knockin the cholera toxin B subunit(ctxb) gene at the lacZ site, then we found that CWD1 can express protein CTB efficiently and CTB was secreted out of the cell. So we assured that the sites of structure genes in the lac operon of Escherichia coli chromosome were suitable for expressing foreign genes .

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