EGFRvⅢ特异性单链抗体的制备及其靶向性能  

Preparation of EGFRvⅢ specific single-chain Fv and its targeting activity

作　　者：张庆丽[1,2] 石必枝[2] 蒋华[2] 周敏[2] 王海[2] 孔娟[2] 谢海龙[1] 

机构地区：[1]南华大学肿瘤研究所,湖南衡阳421001 [2]上海交通大学医学院上海市肿瘤研究所癌基因及相关基因国家重点实验室,上海200032

出　　处：《中国肿瘤生物治疗杂志》2011年第1期11-16,共6页Chinese Journal of Cancer Biotherapy

基　　金：卫生部"重大新药创制"科技重大专项资助项目( No. 2009ZX09103-701)~~

摘　　要：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 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.

关 键 词：EGFRvⅢ SCFV 脑胶质瘤 肝癌 移植瘤 靶向治疗 活体成像 

分 类 号：R392.1[医药卫生—免疫学] R730.51[医药卫生—基础医学]