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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。
Objective: To establish a cell line stably expressing EGFR-GFP fusion protein. Methods: Plasmid pEGFR-EGFP was transformed into DH5α by CaCl 2 method. After digestion and identification, the plasmid was transfected into CHO-K1 cells by electroporation. EGFP was used as fluorescent reporter to clone and culture to obtain monoclonal positive cells. Flow cytometry The expression of EGFR-GFP fusion protein was detected by fluorescence microscopy and Western Blot. The growth curves of transfected cells and untransfected cells were compared. The expression of EGFR-GFP fusion protein Stability. Results: One cell line stably expressing EGFR-GFP fusion protein was obtained and named as CHO-EGFR-GFP1. The fusion protein was mainly expressed on the cell membrane. There was no significant difference in the growth characteristics between stable transfected cells and untransfected cells. CONCLUSION: The successfully obtained cell line expressing EGFR-GFP fusion protein on the membrane surface has laid a foundation for the development of antitumor drugs targeting EGFR and the interaction between EGFR and its ligand.