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-Thrombin-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 for EGFRvⅢ specific single-chain Fv (scFv) by phage display library and to examine its targeting activity. Methods: EGFRvⅢ specific scFv phage library was constructed, and the positive EGFRvⅢ-scFv clone was screened by ELISA. After cloned into pCANTAB-Thrombin-His vector, EGFRvⅢ-scFv plasmid was transformed into E.coli HB2151, and soluble EGFRvⅢ-scFv was induced by IPTG. The specific binding activity of EGFRvⅢ-scFv with EGFRvⅢ was studied by indirect immunofluorescence and in vivo imaging. Results: An EGFRvⅢ-scFv phage library was successfully constructed and 16 EGFRvⅢ-scFv positive clones were identified by ELISA. One clone named EGFRvⅢ-scFv-2A1 was re-cloned into pCANTAB-Thrombin-His vector and soluble EGFRvⅢ-scFv-2A1 was successfully obtained. EGFRvⅢ-scFv-2A1 could specifically bind with HuH7-EGFRvⅢ and HuH7 hepatoma cells, but not with HuH7-EGFR and HuH7 cells in vitro. In vivo, fluorescence-labeled EGFRvⅢ-scFv-2A1 could only bind with U87MG-EGFRvⅢ glioma cells implanted tumor tissues, but not with that of U87MG cells implanted ones. Conclusion: The prepared EGFRvⅢ-scFv-2A1 can specifically bind with EGFRvⅢ, and it might be used for diagnosis and targeted therapy of tumors.

卫生部“重大新药创制”科技重大专项资助项目（No. 2009ZX09103-701）