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目的:构建ureI和ctB-ureI真核表达质粒并分析其在细胞的表达情况。方法:用pIRES-RD2真核表达载体分别在5′端连接ureI和ctB-ureI基因,经双酶切和测序鉴定正确后,相应质粒用脂质体转染HEK293T细胞,用荧光显微镜观察荧光标签,确定质粒的转染率;用人抗幽门螺旋杆菌抗体和马抗CtB抗体,采用免疫荧光和免疫酶组织化学等方法研究该两种真核表达质粒在细胞中目的蛋白表达情况。结果:成功构建了ureI和ctB-ureI的真核表达质粒pIRES-RD2-ureI和pIRES-RD2-ctB-ureI,用此质粒转染HEK293T细胞48～72h后,荧光显微镜显示该两种质粒的转染率约为80%;用免疫荧光和免疫酶组织化学方法表明了该两种基因均能在HEK293T细胞表达并有免疫反应性,表达的ureI主要分布在胞质内或细胞膜附近,ctB-ureI在CtB信号肽引导下分布于HEK293T细胞质、细胞膜附近以及细胞外。结论:成功构建了真核载体pIRES2-DsRed2-ureI和pIRES2-DsRed2-ctB-ureI,目的蛋白表达于HEK293T细胞胞质内并有一定免疫反应性。
Objective: To construct eukaryotic expression plasmids of ureI and ctB-ureI and analyze their expression in cells. Methods: The eukaryotic expression vector pIRES-RD2 was used to ligate the ureI and ctB-ureI genes respectively at the 5 ’end. After double enzyme digestion and sequencing, the corresponding plasmids were transfected into HEK293T cells by lipofectamine. Fluorescence microscopy To determine the transfection efficiency of the plasmid; using human anti-Helicobacter pylori antibody and horse anti-CtB antibody, using immunofluorescence and immunohistochemistry to study the expression of the target protein in the two eukaryotic expression plasmids. Results: The eukaryotic expression plasmids pIRES-RD2-ureI and pIRES-RD2-ctB-ureI of ureI and ctB-ureI were successfully constructed. After transfected HEK293T cells with this plasmid for 48-72h, the expression of these two plasmids The staining rate was about 80%. Immunofluorescence and immunoenzymatic histochemistry showed that both of the two genes were expressed in HEK293T cells and immunoreactive. The expression of ureI mainly distributed in the cytoplasm or in the vicinity of the cell membrane, and ctB-ureI CtB signal peptide distribution in the HEK293T cytoplasm, near the cell membrane and extracellular. CONCLUSION: The eukaryotic vectors pIRES2-DsRed2-ureI and pIRES2-DsRed2-ctB-ureI have been successfully constructed. The target protein is expressed in the cytoplasm of HEK293T cells and has certain immunoreactivity.