可溶性EGF受体胞外域在HeLa细胞中的表达及鉴定

Expression and identification of soluble EGF receptor extracellular domain in HeLa cells

目的:在哺乳动物细胞中表达可溶性人表皮生长因子受体(sEGFR)并进行鉴定。方法:以带信号肽的sEGFR的真核表达载体pEGFR s或pEGFP-N1空载体转染HeLa细胞,利用流式细胞仪和激光共聚焦显微镜分析外源基因的表达水平,并以免疫印迹法分析培养上清中的sEG-FR。结果:转染pEGFP-N1的HeLa细胞,48 h后EGFP的表达百分率可达83%,而转染pEGFR s载体的HeLa细胞EGFP阳性率为4.8%,表明由于插入的sEGFR基因包含终止密码而抑制了EG-FP的表达;共聚焦显微镜观察也得到一致的结果。进一步以抗EGFR结构域L2的抗血清进行免疫印迹分析证明,转染pEGFR s的HeLa细胞培养上清液中表达sEGFR,存在相对分子质量为83 000和80 000两种产物。结论:转染表达载体的HeLa细胞分泌表达sEGFR。

Aim: To express and identify the soluble human epidermal growth factor receptor extracellular domain(sEGFR) in mammalian cells.Methods: HeLa cells were transfected with the expression vector pEGFRs for sEGFR containing signal peptide or with empty vector pEGFP-N1.The expression levels were analyzed with flow cytometry and confocal microscopy and immunoblotting analysis was used to identify sEGFR in the culture supernatant.Results: The EGFP expression rate in HeLa cells transfected with pEGFP-N1 was 83%,wher...