小鼠甘丙肽1型受体在HEK293A细胞中的表达和内化

Expression and Ligand-induced Internalization of EGFP-tagged Mouse Galanin R1 Receptors in HEK293A Cells

目的:克隆小鼠甘丙肽1型受体(mGalR1)基因,构建其真核表达载体,并观察该基因在HEK293A细胞中的表达和内化。方法:应用RT-PCR方法,以小鼠下丘脑RNA为模板,扩增获得甘丙肽I型受体(mGalR1)基因并定向克隆到pEGFP-N1真核表达载体;mGalR1-pEGFP表达载体瞬时转染HEK293A细胞,利用激光共聚焦显微技术观察mGalR1在给予甘丙肽(10-7mol/L)0、5、10、15min刺激时的内化情况。结果:克隆得到正确的mGalR1基因全长序列,其cDNA为1 047bp,共编码348个氨基酸;共聚焦显微镜结果表明mGalR1主要在膜上表达,经甘丙肽刺激5～15min后受体下膜进入胞浆。结论:成功构建真核表达载体mGalR1-pEGFP并在HEK293A中表达;在甘丙肽刺激下小鼠甘丙肽1型受体存在内化现象。

Objective： The study was designed to clone mouse Galanin R1 receptor（mGalR1） gene and analysis its expression and trafficking in HEK293A cells.Method： mGalR1 gene was amplified from RNA which was extracted of mouse hypothalamus by RT-PCR and inserted into an expression vector pEGFP-N1 to get a fusion expression vector named mGalR1-pEGFP.The recombinant expressional vector was transiently transfected into HEK293A cells with LipofectAMINE2000 reagent.Expression and internalization of mGalR1-EGFP was analyzed with confocal laser scanning fluorescence microscopy（Leica SP5） after incubation with galanin（10-7 mol/L and 37℃）.Result： The cloned full-length cDNA sequence of mGalR1 gene was 1047bp,which determined to encode 348 amino acids and showed 100% homology with the published sequence in the GenBank.The mGalR1-EGFP was predominantly localized on the plasma membrane.The membrane expressing mGalR1-EGFP was internalized within 5~15min,with a dramatic reduction in plasma membrane localization and appearance in intracellular vesicles,after incubation of galanin.Conclusion： The recombinant plasmid mGalR1-EGFP was successfully constructed and mGalR1 was expressed and undergone ligand-dependent internalization in HEK293A cells.