构建原核表达质粒pGEX-4T-2-GFP并鉴定其在大肠杆菌中的表达

Construction of pGEX-4T-2-GFP prokaryotic expressing plasmid and its protein expression

目的:构建绿色荧光蛋白(GFP)的原核表达质粒,用以研究小鼠巨噬细胞吞噬细菌的能力。方法:编码GFP的聚合酶链反应(PCR)产物经Eco RⅠ和Bam HⅠ双酶切后,与双酶切后的载体p GEX-4T-2相连,将p GEX-4T-2-GFP转化E.coli DH5α提取质粒,经测序后转化E.coli BL21中诱导GST-GFP融合蛋白表达,将表达融合蛋白的大肠杆菌滴入培养巨噬细胞的皿中,于荧光显微镜下观察GFP的表达及大肠杆菌被吞噬情况。结果:获得的重组质粒测序正确。荧光显微镜下能清晰观察到培养基中及被巨噬细胞吞噬后发绿色荧光的大肠杆菌。结论:成功构建了pGEX-4T-2-GFP重组质粒,为研究小鼠巨噬细胞吞噬细菌的能力创造条件。

Objective： To construct prokaryotic expressing plasmid of GFP gene. Methods： The PCR product of GFP coding sequence, which was digested with Eco R I and Bam H I restriction enzymes, was taped into the plasmid p GEX-4T-2. Then p GEX-4T-2-GFP was transformed into E.coli DH5α and plasmid DNA was extracted. The recombinant plasmid was sequenced and then the expression of GST-GFP fusion protein was induced in BL21. After that, the E.coli which expressed GST-GFP fusion protein was transferred into the dish of cultivating macrophages. Fluorescence microscope was utilized to observe the GFP expression and e.coli swallowed. Results： The recombinant plasmid was sequenced correctly. E. coli expressing GFP both in medium and swallowed by macrophages can be observed clearly with the fluorescence microscope. Conclusion： The recombinant prokaryotic expressing plasmid p GEX-4T-2-GFP was successfully constructed, which facilitated the research for phagocytic capacity of macrophage.