表皮生长因子受体；绿色荧光蛋白；细胞系____

稳定表达EGFRGFP融合蛋白细胞系的建立

http://www.100md.com 《郧阳医学院学报》 2006年第3期

表皮生长因子受体；绿色荧光蛋白；细胞系,,\],表皮生长因子受体；绿色荧光蛋白；细胞系,1材料和方法,2结果,3讨论,[参考文献\]

     [摘要\] 目的：建立稳定表达EGFRGFP融合蛋白的细胞系。方法：CaCl2法将pEGFREGFP质粒转化DH5α，酶切鉴定后提取质粒经电穿孔转染CHOK1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EGFRGFP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EGFRGFP融合蛋白的稳定性。结果：获得1株稳定表达EGFRGFP融合蛋白的细胞系，命名为CHOEGFRGFP1，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EGFRGFP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 EGFRGFP Fusion Protein

    ZHAO Xiao-rong,CAO Li-min,PENG Ji-lin,WANG Min,ZHAO Xiao-ping,WU Sha,LI Wen-han,YE Qing,SHEN Guan-xin.

    (1Department of Immunology,Tongji Medical College,Huazhong University of Science and Technology,Wuhan,Hubei 430030,China;2Department of Epidemiology,School of Public Health,Guangdong Pharmaceutical University,Guangzhou,Guangdong 510310,China;3Department of Immunology,Yunyang Medical College,Shiyan,Hubei 442000,China)

    Abstract: Objective To establish a cell line expressing epidermal growth factor receptor-enhanced green fluorescent protein (EGFRGFP) fusion protein. Methods The E. coli DH5α was transformed with pEGFREGFP plasmid by CaCl2 method. After identified by restriction enzyme digestion, the plasmid was extracted and then transfected into CHOK1 cells by electroporation.A single clone expressing EGFRGFP 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 EGFRGFP fusion protein expression. The growth rate of the transfected and untransfected CHOK1 cells was compared. The transfected cell was frozen, thawed, and passaged to identify stability of the expression of EGFRGFP fusion protein by FCM. Results One cell line expressing EGFRGFP fusion protein was obtained stably and named after CHOEGFRGFP1, EGFRGFP fusion protein was localized mainly in the cell membrane. There was no significant difference between the transfected and untranfected CHOK1 cells on cell growth rate. Conclusion A cell line expressing the EGFRGFP fusion protein stably was established. This work provides a basis for further development of antitumor durgs targeting EGFR and investigation of interactions between EGFR and its ligands. ......

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