S100A13 基因真核表达载体的构建及其在COS-7 细胞中的表达
Construction of Eukaryotic Express Vector with Human S100A13 Gene and Its Expression in COS-7 Cells
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摘要: 目的 构建以绿色荧光蛋白（GFP）为报告基因的重组表达质粒pcDNA3.1/NT-GFP-S100A13,观察S100A13基因在COS-7细胞中的表达及定位。方法 采用克隆和亚克隆技术构建S100A13基因真核表达载体,脂质体介导转染COS-7细胞,RT-PCR和western-blot验证其mRNA和蛋白的表达,荧光显微镜观察该基因亚细胞定位。结果 酶切和测序结果证实重组质粒含有S100A13编码区序列且插入方向正确,转染后观察该基因亚细胞定位于全胞浆。结论 成功构建了S100A13基因真核表达载体,该基因在COS-7细胞中成功表达,S100A13基因表达定位于全胞浆。Abstract: Objective To obtain eukaryotic express vector of human S100A13 gene and detect it s expression in COS-7 cells. Methods S100A13 cDNA was amplified by RT-PCR f rom papillary thyroid carcinoma tissues, Fresh PCR product s was cloned into p GEM-T vector and sequenced. Then the recombinant ORF was subcloned into pcDNA 3. 1/ NT-GFP-topo vector and sequenced. The recombinant vector was transfected into COS-7 cells by lipofectamine, the expression level of S100A13 mRNA and protein were identified by RT-PCR and western2blot, respectively. Results S100A13 gene was identified to be iserted into pcDNA3. 1/ NT-GFP-topovector correctly and the recombinant vector expressed S100A13 mRNA and protein stably. The green fluorescence protein was expressed in the whole cytoplasm in COS-7 cells. Conclusion The eukaryotic express vector containing human S100A13 gene was successfully const ructed and expressed. The subcellular localization of S100A13 is in the whole cytoplasm in COS-7 cells .
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