真核表达载体pEGFP-N2/AT2R的构建及其在原代血管平滑肌细胞的表达

Construction of eukaryotic expressing vector pEGFP-N2/AT2R and its expression in primary vascular smooth muscle cells

目的体外构建携带血管紧张素Ⅱ2型受体(ANGⅡType 2 Receptor,AT2R)基因的增强型绿色荧光蛋白真核表达载体,并观察其在大鼠血管平滑肌细胞(vascular smooth muscle cells,VSMCs)中的表达。方法以pUHD-10.3/AT2R质粒为模板,PCR扩增AT2R基因的全长cDNA序列,再将其克隆入载体pEGFP-N2,构建其真核表达载体pEGFP-N2/AT2R。以基因转染技术,将AT2R导入原代VSMCs。倒置荧光显微镜观测转染后VSMCs生长变化及AT2R在其中的表达等情况。图像分析技术检测AT2R在VSMCs中的转染效率。Western blot检测转染AT2R基因的VSMCs表达其编码蛋白。RT-PCR法对AT2R基因修饰的VSMCs进行鉴定。结果成功构建AT2R基因的真核表达载体pEGFP-N2/AT2R,该真核载体能携带AT2R基因转染并有效表达于VSMCs,其转染效率约40%,RT-PCR及Western blot均可检测到AT2RmRNA及蛋白在血管平滑肌细胞中高表达。结论成功地将克隆到的AT2R基因克隆入pEGFP-N2载体中,并实现了AT2R基因在原代VSMCs的表达。

Objective To clone Ang Ⅱ type 2 receptor（AT2R） gene,obtain enhanced green fluorescent protein（EGFP） vector for the gene,and then observe its expression in primary vascular smooth muscle cells（VSMCs） after transfection.Methods Utilizing plasmid pUHD-10.3/AT2R as a template,the full length cDNA fragment of AT2R was amplified by polymerase chain reaction（PCR）.Eukaryotic expression vector pEGFP-N2/AT2R was constructed by cloning AT2R gene into vector pEGFP-N2.After transfecting AT2R into primarily cultured VSMCs,invert fluorescent microscopy was employed to observe VSMCs growth as well as AT2R expression in VSMCs.Transfection efficiency of AT2R in VSMCs was evaluated by image analyzor,the protein and mRNA expressions of AT2R in VSMCs were examined by Western blotting and RT-PCR.Results Eukaryotic expression vector pEGFP-N2/AT2R was successfully constructed.The eukaryotic vector expressing AT2R gene was efficiently transfected and expressed in VSMCs.The transfection efficiency of AT2R gene in VSMCs was about 40%.RT-PCR and Western blotting indicated that AT2R was expressed in cultured VSMCs at mRNA and protein levels.Conclusion cDNA sequence encoding AT2R gene is successfully cloned into pEGFP-N2 which can be efficiently expressed in primary VSMCs.