含绿色荧光蛋白和小鼠T—bet基因双顺反子真核表达载体的构建及其在A549细胞中的表达

Construction of bi-cistronic eukaryotic vector carrying green fluorescen protein and murine T-bet gene and its expression in AS49 cells

目的构建一种带有绿色荧光蛋白（GFP）和小鼠T—bet基因双顺反子的新型真核表达载体pCI—mT—bet—IRES—GFP。方法利用脑炎心肌炎病毒（EMCV）的内部核糖体进入位点（IRES），将GFP的cDNA与小鼠T—bet基因（mT—bet）连接到真核表达载体pCI中，构建含GFP和mT—bet基因双顺反子的重组质粒。结果成功构建了含GFP和mT—bet基因双顺反子的真核表达载体。荧光显微镜下可观察到发绿色荧光的转基因细胞。结论含绿色荧光蛋白和mT—bet基因双顺反子真核表达载体的构建为哮喘基因治疗研究提供了新的生物表达体系。

Objective To generate a bi-cistronic eukaryotic vector coexpressing murine T-bet and GFP for facilitating the study of T-bet gene therapy. Methods A 600 bp IRES from encephalomyocarditis virus and 700 bp GFP were trimolecularly ligated into EcoR I site of eukaryotic vector pCI. An approximately 1.7 kb murine T-bet cDNA segment was then ligated into Xho I site of pCI. Result The bi-cistronic plasmids coexpressing murine T-bet and GFP was identified by restriction enzymes map. Conclusion The murine T-bet cDNA eukaryotic expression vector pCI-mT-bet-GFP is successfully constructed, which can provide a new expressive system for the research of gene therapy in asthma.