人GFP-AWP1融合基因载体的构建及其在293细胞中的表达

目的 克隆、构建绿色荧光蛋白(GFP)-AWP1(associated with protein kinase C related kinase 1,AWP1)表达载体,观察AWP1在293细胞中表达和定位。方法 采用逆转录PCR(RT-PCR)法从人ECV304内皮细胞中扩增AWP1cDNA编码区,并将其重组于GFP表达载体pEGFP-C2中。经酶切、序列鉴定分析后,将该重组质粒通过DOTAP脂质体介导,转染293细胞。荧光显微镜观察AWP1在细胞内的表达和分布。结果 GFP-AWP1融合基因表达载体经酶切鉴定和测序分析确认构建成功,并在293细胞中获得了高效表达。荧光显微镜下,在不携带外源基因的空载体pEGFP-C2转染的对照组293细胞,绿色荧光均匀分布于整个细胞中;在重组质粒pEGFP-C2/AWP1转染的293细胞,绿色荧光弥散分布于细胞质内。结论 成功构建GFP-AWP1融合基因表达载体并表达于293细胞胞质中。

Construction of GFP-AWP1 fusion gene vector and its expression in 293 cells

Objective To construct green fluorescent protein (GFP)-AWP1 (a novel human protein associated with protein kinase C-related kinase 1) fusion gene vector for observing the expression and localization of AWP1 in 293 cells. Methods The coding region in AWP1 cDNA was amplified by RT-PCR from human endothelial cell line ECV304 and recombined into pEGFP-C2 plasmid expressing GFP. After identification with restriction endonucleases and sequence analysis, the recombinant plasmid was transfected into 293 cells with the cationic liposome DOTAP as the transfection reagent. The expression and localization of AWP1 were observed under a fluorescence microscope. Results Restriction endonuclease assay and sequence analysis verified the successful construction of the recombinant vector pEGFP-C2/AWP1, and GFP-AWP1 fusion protein was highly efficiently expressed in 293 cells. Under fluorescent microscope, green fluorescence was seen homogeneously distributed in the entire cell body of the cells transfected by the empty vector pEGFP-C2, but diffusely in the cytoplasm of the cells transfected by the recombinant vector pEGFP-C2/AWP1. Conclusion GFP-AWP1 fusion gene vector is successfully constructed and the fusion protein expressed in the cytoplasm of 293 cells.