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Preparation of EGFRvⅢ specific single-chain Fv and its targeting activity
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Abstract:
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.
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Project Supported:
Project supported by the Major Science and Technology Special Program for “Significant New Drug Creation” of Ministry of Health (No. 2009ZX09103-701)
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