神经特异性转录因子DAT1绿荧光蛋白表达载体的构建及其在C8细胞中的表达定位

目的：构建神经特异性转录因子DAT1与绿荧光蛋白融合表达的表达载体，并检测其在C8星形胶质细胞中的表达。方法：采用基因重组技术将DAT1基因和绿色荧光蛋白基因融合，构建绿荧光蛋白表达载体pEGFP—C1-DAT1，测序验证序列无误后。用脂质体法将重组质粒转入C8细胞，荧光显微镜观察DAT1的表达及定位。结果：测序验证结果表明，重组质粒含有DAT1全长编码序列，转染实验表明EGFP—DAT1能在C8细胞中正确表达，且：DAT1蛋白主要定位于细胞核中。结论：DAT1全长cDNA基因绿色荧光蛋白表达载体构建成功，在C8细胞中可以正确表达，为进一步研究DAT1的功能奠定了基础。

Construction of recombinant DAT1/EGFP vectors and its expression in C8 cells

Objective:To construct the fusion gene of DAT1 with green flurescent protein(GFP),and study its expression in C8 cells. Methods: DAT1 gene was ligated into the multiple clone site of pEGFP-C1 vector by recombination technique. After confirmation with sequence analysis, the expression vector pEGFP-C1-DAT1 was transformed into C8 cells by Lipofectamin 2000. The protein expression was observed by fluorescence microscope. Results: DNA sequence analysis showed that DAT1 cDNA had been successfully inserted into pEGFP-C1 expression vector. The C8 cells transfected with pEGFP-C1-DAT1 could be detected expressing GFP-DAT1. Conclusion: The eukaryotic expression vector containing DAT1 is successfully constructed and expressed.