人G蛋白偶联受体107表达载体构建及细胞定位的研究

Construction and cellular localization of human G protein coupled receptor 107 expression vector

目的克隆人G蛋白偶联受体107(GPR107)基因以构建真核表达载体,并探讨GPR107在真核细胞中的亚细胞定位。方法通过分子克隆技术克隆人GPR107基因的cDNA;利用真核细胞表达载体pCMV-Tag2B构建重组载体pCMV-Tag2B-GPR107,瞬时转染至293T细胞,Western blot检测GPR107的表达变化;采用免疫荧光标记,激光共聚焦显微镜观察GPR107在细胞中的定位。结果经过基因测序证实获得人GPR107的cDNA;成功构建真核表达载体pCMV-Tag2B-GPR107;Western blot结果显示,与未转染组比较,转染组GPR107表达量明显上调。激光共聚焦显微镜观察显示GPR107主要表达于细胞质。结论GPR107在293T细胞中过表达,并定位于细胞质。

Objective To construct eukaryotic expression vector and to explore the subcellular localization of GPR107 in eukaryotic cells,human G protein coupled receptor 107(GPR107)gene is cloned.Methods Firstly,the cDNA of human GPR107 gene was cloned by molecular cloning technique,and the recombinant vector pCMV-Tag2B-GPR107 was constructed by eukaryotic expression vector pCMV-Tag2B.The expression of GPR107 was detected by Western blot,and the localization of GPR107 in 293T cells was observed by immunofluorescence labeling and laser confocal microscope.Results The cDNA of human GPR107 was confirmed by gene sequencing and the eukaryotic expression vector pCMV-Tag2B-GPR107 was constructed successfully.The results of Western blot showed that compared with the non-transfection group,the expression of GPR107 in the transfection group was significantly up-regulated.Laser confocal microscopy showed that GPR107 was mainly expressed in the cytoplasm.Conclusion GPR107 is overexpressed in 293T cells and localized in the cytoplasm.