稳定表达EGFR.DOCVIP

临床医学论文-稳定表达EGFR ?????????????? 作者：赵晓蓉，曹利民，彭吉林，王敏，赵晓平，吴砂，李文涵，叶庆， 沈关心? [摘? 要]? 目的：建立稳定表达EGFRGFP融合蛋白的细胞系。方法：CaCl2法将pEGFREGFP质粒转化DH5α，酶切鉴定后提取质粒经电穿孔转染CHOK1细胞；以EGFP作为荧光报告分子，克隆化培养以获取单克隆阳性细胞；流式细胞术、荧光显微镜术、Western Blot检测转染细胞EGFRGFP融合蛋白的表达；比较稳定转染细胞及未转染细胞的生长曲线；反复冻存、复苏、传代鉴定细胞表达EGFRGFP融合蛋白的稳定性。结果：获得1株稳定表达EGFRGFP融合蛋白的细胞系，命名为CHOEGFRGFP1，融合蛋白主要表达于细胞膜。稳定转染细胞和未转染细胞生长特性无显著差异。结论：成功获得膜表面稳定表达EGFRGFP融合蛋白的细胞系，为研制以EGFR为靶点的抗肿瘤药物以及研究EGFR与其配体间相互作用奠定了基础。 　　[关键词]? 表皮生长因子受体；绿色荧光蛋白；细胞系 　　Establishment of a Cell Line Expressing EGFRGFP Fusion Protein??? 　　Abstract: Objective To establish a cell line expressing epidermal growth factor receptor-enhanced green fluorescent protein (EGFRGFP) fusion protein. Methods The E. coli DH5α? was transformed with pEGFREGFP plasmid by CaCl2 method. After identified by restriction enzyme digestion, the plasmid was extracted and then transfected into CHOK1 cells by electroporation.A single clone expressing EGFRGFP fusion protein was obtained by seeding the cells into 96-well plates with one cell per well. GFP-positive clones were detected by flow cytometry, inverted fluorescence microscopy, and western blot analysis for EGFRGFP fusion protein expression. The growth rate of the transfected and untransfected CHOK1 cells was compared. The transfected cell was frozen, thawed, and passaged to identify stability of the expression of EGFRGFP fusion protein by FCM. Results One cell line expressing EGFRGFP fusion protein was obtained stably and named after CHOEGFRGFP1, EGFRGFP fusion protein was localized mainly in the cell membrane. There was no significant difference between the transfected and untranfected CHOK1 cells on cell growth rate. Conclusion A cell line expressing the EGFRGFP fusion protein stably was established. This work provides a basis for further development of antitumor durgs targeting EGFR and investigation of interactions between EGFR and its ligands. 　　Key words: Epidermal growth factor receptor; EGFP; Cell line 　　表皮生长因子受体（epidermal growth factor recep