【摘要】 目的:分别构建含有樱桃红荧光蛋白和蓝色荧光蛋白(BFP)标签的应激蛋白G3BP表达质粒,实现活细胞内G3BP蛋白的红/蓝色荧光标记。方法:以重组质粒pEGFP-C1-G3BP为模板,PCR法扩增出目的基因,分别利用EcoRI和BamHI双酶切法将目的片段连接到pmCheery-C1和pEBFP-N1载体上,再将构建成功的pmCheery-C1-G3BP和pEBFP-N1-G3BP重组质粒转染入HeLa细胞内,以激光共聚焦显微镜及Western印迹法检测红/蓝色荧光蛋白与目的蛋白G3BP的融合表达以及在活细胞内的共定位情况。结果:以单/双酶切及基因测序法鉴定构建的重组质粒均无误,荧光显微镜及Western印迹结果均检测到红/蓝色融合蛋白的表达,共定位分析结果显示两者均可在HeLa细胞胞浆中形成应激颗粒。结论:pmCheery-C1-G3BP和pEBFP-N1-G3BP构建成功,对G3BP蛋白的红/蓝色荧光标记有助于进行其在细胞应激方面的机制研究。更多还原

【Abstract】 Objective: To construct eukaryotic cheery red fluorescent protein /blue fluorescent protein(BFP) expressing recombinant plasmids of G3BP(Ras-GTPase activating protein SH3 domain binding protein),pmCheery-C1-G3BP and pEBFP-N1-G3BP.Methods: The genes of G3BP were amplified by PCR from the recombinant pEGFP-C1-G3BP plasmid and inserted into pmCheery-C1 or pEBFP-N1 fluorescent expressing vector with EcoRI and BamHI site.The two recombinant plasmids were transfected into HeLa cells and the expression of red/blue fluorescent fusion proteins was examined by fluorescence microscope and western blotting assay.Results: The recombinant plasmids were sequenced correctly and detected in the products of the restriction single/double enzyme digestion.The red or blue fluorescent fusion G3BP proteins were also detected to form the stress granules in the cytoplasm of transfected HeLa cells by fluorescence microscope and western blotting assay.Conclusion: These recombinant eukaryotic plasmids of pmCheery-C1-G3BP and pEBFP-N1-G3BP are constructed successfully and that helps to study the role of G3BP protein in cellular stress granules formation.更多还原