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目的克隆、构建绿色荧光蛋白(GFP)-AWP1(associatedwithproteinkinaseCrelatedkinase1,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。
Objective To clone and construct the expression vector of green fluorescent protein (AWP1) associated with protein kinase (AWP1), and to observe the expression and localization of AWP1 in 293 cells. Methods The coding region of AWP1 cDNA was amplified from human ECV304 endothelial cells by reverse transcription polymerase chain reaction (RT-PCR) and recombined in GFP expression vector pEGFP-C2. After digestion and sequence analysis, the recombinant plasmid was transfected into 293 cells through DOTAP liposome. Fluorescence microscopy was used to observe the expression and distribution of AWP1 in cells. Results The GFP-AWP1 fusion gene expression vector was confirmed by restriction analysis and sequencing analysis, and was successfully constructed and highly expressed in 293 cells. Under the fluorescence microscope, the green fluorescence of 293 cells transfected with empty vector pEGFP-C2 without exogenous gene was evenly distributed in the whole cells. In 293 cells transfected with recombinant plasmid pEGFP-C2 / AWP1, Distributed in the cytoplasm. Conclusion The GFP-AWP1 fusion gene expression vector was successfully constructed and expressed in the cytoplasm of 293 cells.