P21-GFP融合基因表达载体的构建与在视网膜色素上皮细胞的表达定位

目的构建绿色荧光蛋白GFP—p21融合基因表达载体，并观察其在人视网膜色素上皮细胞中的表达及定位，为研究细胞周期蛋白激酶抑制因子p21对视网膜色素上皮细胞细胞周期的影响提供实验基础。方法以含有p21的全长序列的质粒为模板PCR扩增p21 cDNA，应用基因重组技术与GFP基因融合，构建以绿色荧光蛋白GFP为报告基因的重组表达质粒pGFP—p21。利用脂质体转染体外培养的人视网膜色素上皮细胞，在活细胞状态下用荧光显微镜直接观察pGFP—p21融合蛋白在细胞中的分布和定位，用Western Blot方法分析其蛋白的表达。结果①酶切、DNA测序均证实插入片断的正确性。②荧光显微镜观察结果显示在空载体pEGFP—C1转染组中，细胞内绿色荧光呈弥散分布；重组质粒pGFP—p21转染组中，绿色荧光主要集中在细胞核内。③Western Blot证实了pGFP—P21融合基因的表达。结论成功的构建了pGFP—p21质粒。视网膜色素上皮细胞能高效表达pGFP—p21融合基因，且主要定位于细胞核内，与p21基因的表达方式相同。

Construction of P21/EGFP fusion gene expression vector and its expression in hRPE cells

Objective To construct eukaryotic expression vector P21/EGFP ,which can express GFP-P21 fusion protein, and to investigate its expression in hRPE. Methods human P21 ORF was amplified from pCEP-P21 by PCR and inserted into plasmid pEGFP-C1.Using lipofectin method,the recombinant expression plasmid pEGFP-P21 was transfected into hRPE cells. Expresstion product of GFP-P21 fusion protein was analyzed by Western- blotting and observed localization under fluorescent microscope. Results eukaryotic expression vector pEGFP-P21 was constructed successfully and GFP-P21 fusion protein were expressed ideally in hRPE and localized in nuclei. Conclusion GFP-P21 fusion protein immediately expressed in hRPE successfully and localized in nuclei. The use of GFP for cell marking in gene P21 demonstrated a new technology for functional study of gene P21.