稳定表达EGFR-GFP融合蛋白细胞系的建立

目的:建立稳定表达EGFR-GFP融合蛋白的细胞系。方法:CaC l2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、W estern B lot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。

Establishment of a Cell Line Expressing EGFR-GFP Fusion Protein

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 CaCl_2 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.