上海交通大学学报（医学版）

›› 2012, Vol. 32 ›› Issue (11): 1406-.doi: 10.3969/j.issn.1674-8115.2012.11.003

• 专题报道（病原微生物学） • 上一篇    下一篇

大肠埃希菌中λ-Red同源重组系统介导的trp和yfp基因融合表达的研究

陈 兴1, 姚玉峰2, 周爱萍2, 倪进婧2   

  1. 1.华中农业大学工学院, 武汉 430070； 2.上海交通大学 基础医学院病原生物学教研室, 上海 200025
  • 出版日期:2012-11-28 发布日期:2012-11-30
  • 通讯作者: 倪进婧, 电子信箱: nijinjing45@163.com。
  • 作者简介:陈 兴（1987—）, 男, 硕士生；电子信箱: chenxing3753@qq.com。
  • 基金资助:

    国家自然科学基金（30870100）

Gene fusion and expression of trpA and yfp mediated by λ-Red recombinant system in Escherichia coli

CHEN Xing1, YAO Yu-feng2, ZHOU Ai-ping2, NI Jin-jing2   

  1. 1.College of Engineering &|Technology, Huazhong Agricultural University, Wuhan 430070, China；2.Department of Medical Microbiology and Parasitology, Shanghai Jiaotong University, Shanghai 200025, China
  • Online:2012-11-28 Published:2012-11-30
  • Supported by:

    National Natural Science Foundation of China, 30870100

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摘要：

目的 为研究大肠埃希菌中色氨酸合成酶α亚基编码基因(trpA)的表达，利用λ-Red系统将trpA、黄色荧光蛋白基因(yfp)、氯霉素抗性编码基因(cat)融合片段敲入基因组中。方法 将trpA、yfp、cat基因进行融合PCR，获得重组片段，转化入MG1655菌株中，利用pKD46编码的λ-Red系统将片段重组入基因组中，经PCR验证及测序鉴定正确后，荧光显微镜观察融合蛋白表达。结果 成功获得trpA-yfp-cat重组片段，转化入MG1655菌株中获得阳性克隆，经测序鉴定正确。荧光显微镜观察可见细菌胞质内弥散分布黄色荧光，表明trpAyfp融合表达片段成功地在MG1655菌株中表达。结论 运用λ-Red系统可将trpA-yfp融合片段敲入MG1655菌株基因组中原位替换trpA基因，通过观察YFP蛋白的表达间接反映trpA基因的表达，具有不改变trpA基因自身功能和细菌生长的优点，为进一步研究大肠埃希菌色氨酸合成途径奠定基础。

关键词: λ-Red系统, 融合PCR, 黄色荧光蛋白, 色氨酸合成酶α亚基

Abstract:

Objective To investigate the expression of tryptophan synthase α subunit-encoding gene (trpA) of Escherichia coli (E. coli), fusion fragment of trpA, yellow fluorescent protein gene (yfp) and chloramphenicol-encoding gene (cat) was knocked into E.coli strain MG1655 genome using λ-Red system. Methods Target genes trpA, yfp and cat were amplified by PCR, and the recombinant fragment was obtained by fusion PCR. The recombinant fragment was transformed into E.coli strain MG1655 harboring genes of λ-Red system in plasmid pKD46. After PCR checking and sequencing confirmation, the fusion protein expression was observed by fluorescence microscopy. Results The recombinant fragment trpA-yfp-cat was successfully obtained and transformed into MG1655, and the positive clones were screened out and confirmed by sequencing. The yellow fluorescence was observed throughout the bacterial cytoplasm, indicating that trpA-yfp fusion protein was successfully expressed in MG1655. Conclusion The trpA gene could be replaced by trpA-yfp fusion fragment in situ using λ-Red system, and its expression could be reflected indirectly by observing the expression of yellow fluorescent protein. The function of trpA and bacterial growth have not been changed, which lays foundation for the further study of E.coli tryptophan synthesis.

Key words: λ-Red system, fusion PCR, yellow fluorescent protein, tryptophan synthase &alpha, subunit