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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。
Objective To construct recombinant Listeria monocytogenes expressing green fluorescent protein (GFP), which provides an important tool for the study of Listeria monocytogenes. Methods The plasmids of Listeria monocytogenes (phly) and GFP were fused by SOEing PCR and ligated into pCW plasmid to construct recombinant prokaryotic expression plasmid pCW-phly-GFP. The recombinant plasmid was electrotransformed into Listeria monocytogenes, and the fluorescence expression was analyzed by fluorescence microscopy. Listeria monocytogenes carrying the recombinant plasmids were serially subcultured in the BHI broth containing erythromycin and erythromycin, respectively, and the recombinant plasmids pCW-phly-GFP and GFP were isolated from sheep liss Stability in bacteria. Results The recombinant plasmid pCW-phly-GFP was confirmed by enzyme digestion and sequencing. Listeria monocytogenes carrying a recombinant plasmid can be seen under a fluorescence microscope as green fluorescence. Recombinant plasmid pCW-phly-GFP was stably present in Listeria monocytogenes with high expression of GFP under erythromycin resistance stress. Conclusion The prokaryotic expression plasmid pCW-phly-GFP of GFP gene was successfully constructed, which can express GFP efficiently in Listeria monocytogenes. It provides an important tool for further study on Listeria monocytogenes as a vaccine vector.