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目的:应用噬菌体展示技术筛选出能与EGFRvⅢ特异性结合的单链抗体(single-chain Fv,scFv),并研究其靶向性能。方法:构建EGFRvⅢ特异性scFv噬菌体库,ELISA筛选阳性克隆,阳性EGFRvⅢ-scFv质粒重新克隆入pCANTAB-Throm-bin-His载体,转化E.coli HB2151,IPTG诱导可溶性EGFRvⅢ-scFv表达。间接免疫荧光及裸鼠活体成像技术鉴定EGFRvⅢ-scFv与EGFRvⅢ的特异性结合。结果:成功构建了EGFRvⅢ-scFv噬菌体库,ELISA筛选得到16个EGFRvⅢ-scFv克隆,取一克隆命名为EGFRvⅢ-scFv-2A1。EGFRvⅢ-scFv-2A1质粒重新克隆入pCANTAB-Thrombin-His载体,成功表达可溶性EGFRvⅢ-scFv-2A1。EGFRvⅢ-scFv-2A1在体外可特异性结合HuH7-EGFRvⅢ肝癌细胞,但不结合HuH7-EGFR和HuH7肝癌细胞;荧光标记的EGFRvⅢ-scFv-2A1裸鼠体内可特异性结合U87MG-EGFRvⅢ胶质瘤细胞移植瘤,而不结合U87MG细胞移植瘤。结论:成功制备的EGFRvⅢ-scFv-2A1可特异性靶向结合EGFRvⅢ,在肿瘤的诊断和靶向治疗中具有潜在应用价值。
OBJECTIVE: To screen single-chain Fv (scFv) specific binding to EGFRvⅢ by phage display technique and to study its targeting properties. Methods: EGFRvⅢ specific scFv phage library was constructed. The positive clones were screened by ELISA. The positive EGFRvⅢ-scFv plasmid was cloned into pCANTAB-Throm-bin-His vector and transformed into E.coli HB2151. IPTG induced soluble EGFRvⅢ-scFv expression. Indirect immunofluorescence and in vivo imaging of nude mice were used to identify the specific binding of EGFRvⅢ-scFv to EGFRvⅢ. Results: EGFRvⅢ-scFv phage library was successfully constructed and 16 EGFRvⅢ-scFv clones were screened by ELISA. One clone was named EGFRvⅢ-scFv-2A1. EGFRvⅢ-scFv-2A1 plasmid was cloned into pCANTAB-Thrombin-His vector and successfully expressed soluble EGFRvⅢ-scFv-2A1. EGFRvⅢ-scFv-2A1 can specifically bind to HuH7-EGFRvⅢ hepatoma cells in vitro, but not to HuH7-EGFR and HuH7 hepatoma cells. Fluorescently labeled EGFRvⅢ-scFv-2A1 can specifically bind to U87MG-EGFRvⅢ glioma cells Xenografts, but not U87MG cell xenografts. CONCLUSION: The successfully prepared EGFRvⅢ-scFv-2A1 can specifically bind to EGFRvⅢ and has potential value in the diagnosis and targeted therapy of tumors.