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Study of Identfiying EGFRvⅢ Specific Single-chain Fragments Variable and Its Biological Activity

Author: ZhangQingLi
Tutor: XieHaiLong
School: Nanhua University
Course: Pathology and Pathophysiology
Keywords: EGFRv III scFv Targeted therapy Vivo imaging
CLC: R392
Type: Master's thesis
Year: 2011
Downloads: 15
Quote: 0
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Abstract


Objective: The phage display filter out specific binding with EGFRv III single-chain antibody ( single - Chain Fv , scFv ) , and its biological activity . Methods: the use of antigen the PEP3 - KLH immune BALB / c mice , the amplified VH and VL fragment spliced ??into the scFv fragment and cloned into the pCANTAB5E - thrombin vector , electrically transforming HPD3 cells , specific scFv phage library building in EGFRv III . ELISA screening PEP3 - BSA , the analysis of positive clones were sequenced , and a positive EGFRv III - scFv plasmid re - cloning into the pCANTAB - Thrombin - His vector , transformed into E. coli ER2738 the IPTG induction EGFRv III - scFv expression , the supernatant was precipitated by Ni Sepharose 4 Fast Flow purified soluble scFv . Indirect immunofluorescence and in vivo imaging technology identification EGFRv III - scFv with EGFRv Ⅲ specificity of binding . Results: build a storage capacity of 2.23 × 10 8 of EGFRv Ⅲ-scFv phage antibody library ELISA screening 16 EGFRv III - scFv clone , a clone was named for EGFRv III - scFv - 2A1 . Re - cloned into plasmid EGFRv Ⅲ-scFv-2A1 pCANTAB-Thrombin-His vector successfully expressed in soluble EGFRv III - scFv - 2A1 . The EGFRv Ⅲ-scFv-2A1 in vitro specificity combined HuH7 - EGFRv Ⅲ cells , but not combined with HuH7 - EGFR cells ; fluorescently labeled EGFRv Ⅲ-scFv-2A1 in vivo can be enriched to U87MG - EGFRv III cells transplanted tumor , in U87MG cells transplanted tumor at the no enrichment . Conclusion: The screening anti - EGFRv Ⅲ-scFv-2A1, the antibody may play a role in tumor diagnostic imaging and targeted therapy .

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