NHERF4真核表达质粒的构建及其在Caco2细胞中的表达

Observation of constructed NHERF4 eukaryotic expression plasmid in Caco2 cells

目的:构建人NHERF4真核表达质粒,转染人结肠癌Caco2细胞,建立能稳定携带NHERF4基因并高表达的Caco2细胞株,为后期相关实验奠定基础。方法:利用真核表达载体pIRES2-ZsGreen1构建人NHERF4cDNA真核表达质粒,脂质体介导质粒pIRES2-ZsGreen1和NHERF4表达质粒pIRES2-ZsGreen1-NHERF4转染人结肠癌细胞Caco2,荧光显微镜观察判断转染结果。G418筛选稳转细胞株,共聚焦免疫荧光观察NHERF4的亚细胞定位表达,蛋白免疫印迹实验观察NHERF4蛋白表达。结果:荧光显微镜观察证实质粒pIRES2-ZsGreen1和NHERF4表达质粒pIRES2-ZsGreen1-NHERF4均成功转染Caco2细胞。蛋白免疫印迹实验证实转染pIRES2-ZsGreen1-NHERF4质粒的Caco2细胞高表达NHERF4蛋白。结论:成功建立稳定表达外源NHERF4蛋白的pIRES2-ZsGreen1-NHERF4Caco2细胞株,为能进一步分析研究NHERF4与MPR2在结肠癌多药耐药机制中的作用奠定了前期实验基础。

Objective： To construct the recombinant eukaryotic NHERF4 expression plasmid and identify the expression and localization of NHERF4 in Caco2 cells,establishing the foundation for the forthcoming research.Methods：The eukaryotic expressing pIRES2-ZsGreen1 plasmid was chosen as the vector for constructing recombinant pIRES2-ZsGreen1 NHERF4 plasmid.The plasmid was transfected into Caco2 cells by Lipofectamine？2000,G418 screening was followed.Confocal immunofluorescence microscopy was performed for observing the localization of NHERF4 protein.The expression of NHERF4 protein was detected by Western blot.Results：The success of pIRES2-ZsGreen1 and recombinant pIRES2-ZsGreen1-NHERF4 plasmid transfecting into Caco2 cells was confirmed by fluorescence microscope.Western blot proved the higher expression of NHERF4 in pIRES2-ZsGreen1-NHERF4 Caco2 cells.Conclusion：The eukaryotic expression plasmid carrying NHERF4 gene has been successfully constructed and pIRES2-ZsGreen1-NHERF4 Caco2 cells maintained higher NHERF4 protein,and lay the foundation of the preliminary experiment for the further analyze the role of NHERF4 and MPR2 in the mechanism of multidrug resistance of colon cancer.