张祥, 苏琳, 刘思静, 等.表达绿色荧光蛋白的重组绵羊李斯特菌的构建及荧光分析.四川大学学报(医学版),2017,48(6):809-813
表达绿色荧光蛋白的重组绵羊李斯特菌的构建及荧光分析
Construction and Fluorescence Analysis of the Recombinant Listeria ivanovii Strain Expressing Green Fluorescent Protein
  
中文关键词:  绵羊李斯特菌绿色荧光蛋白原核表达
英文关键词:Listeria ivanoviiGreen fluorecent proteinProkaryotic expression
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中文摘要:
      目的 构建表达绿色荧光蛋白(GFP)的重组绵羊李斯特菌,为绵羊李斯特菌的研究提供重要工具。方法 利用SOEing PCR的方法将单核细胞增生性李斯特菌溶血素的启动子(phly)与GFP基因融合,连接到pCW质粒上,构建重组原核表达质粒pCW-phly-GFP。将重组质粒电转化绵羊李斯特菌,利用荧光显微镜分析荧光表达情况。分别在含红霉素和不含红霉素的BHI肉汤中连续传代培养携带重组质粒的绵羊李斯特菌,通过提取质粒和观察荧光的方法研究重组质粒pCW-phly-GFP及GFP在绵羊李斯特菌中的稳定性。结果 重组质粒pCW-phly-GFP酶切验证和测序均正确。在荧光显微镜下可以见到携带重组质粒的绵羊李斯特菌发绿色荧光。在红霉素抗性压力选择下重组质粒pCW-phly-GFP能稳定存在于绵羊李斯特菌中,并高效表达GFP。结论 成功构建了GFP基因原核表达质粒pCW-phly-GFP,能在绵羊李斯特菌中高效表达GFP,为进一步研究绵羊李斯特菌作为疫苗载体提供了重要的工具。
英文摘要:
      ObjectiveConstructing the recombinant Listeria ivanovii strain expressing green fluorescent protein to provide an important tool for study of Listeria ivanovii. MethodsThe promoter of Listeria monocytogenes Listeriolysin O (phly) and the green fluorescent protein (GFP) gene were fused by SOEing PCR, and then ligated the fusion gene into plasmid pCW to result in recombinant plasmid pCW-phly-GFP. Recombinant plasmid was electroporated into Listeria ivanovii, and fluorescence microscope was used to analyze the expression of GFP. To observe the stability of recombinant plasmid and the stable expression of GFP in Listeria ivanovii, bacteria were cultured in the BHI broth with or without erythromycin for several generations. The stability of recombinant plasmid pCW-phly-GFP and fluorescent protein in each generation of bacteriawas studied by extracting plasmids and observing fluorescence. ResultsThe exactness of recombinant plasmid pCW-phly-GFP was confirmed with restrictive endonuclease assay and sequence analysis. Under the fluorescence microscope, the green fluorescence was obvious in Listeria ivanovii carried with pCW-phly-GFP. The recombinant plasmid pCW-phly-GFP was stable in Listeria ivanovii and the GFP kept expressing in a high level under the pressure of erythromycin. ConclusionThe prokaryotic expression plasmid pCW-phly-GFP containing GFP gene was successfully constructed. Listeria ivanovii carried with the plasmid efficiently expressed GFP. This research provides an important tool for further study of Listeria ivanovii as a vaccine carrier.
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