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" Construction, expression and biological activity evaluation of a single- chain Fv antibody frament specific for domain II of HER2 receptor in Eshcherichia coli "
/وجیهه اکبری
; Daryoush Abedi, Hamid Mir Mohammad Sadeghi, Abbas Jafarian Dehkordi, Perry Chou
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Isfahan University of Medical Sciences
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| Document Type
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Latin Dissertation
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| Language of Document
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English
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| Record Number
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103539
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| Doc. No
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T15332
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QW,525,A313c,2014
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| Main Entry
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Akbari, Vajihe
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| Title & Author
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Construction, expression and biological activity evaluation of a single- chain Fv antibody frament specific for domain II of HER2 receptor in Eshcherichia coli/وجیهه اکبری
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| College
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Schools, Pharmacy
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| Date
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, 2014
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| Degree
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Pharmaceutical Biotechnology, Ph.D
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XIV, 126p.: ill, diag, tab
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| Note
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This thesis is a research project with project ID 189113
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| Abstract
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Human epidermal growth factor receptor (HER) family plays an important role in majority types of cancers. Receptor dimerization is necessary for the HER signal transduction pathway and tyrosine kinase activity. Recently, several monoclonal antibodies have been developed to directly interfere with ligand-HER receptor binding and receptor dimerization. A single chain variable fragment (scFv) is a valuable alternative to an intact antibody. A novel monoclonal antibody, pertuzumab, can bind to domain II of HER2 and sterically block its dimerization with other HER members, suggesting that the presence of the Fc region is not necessary for growth inhibition of tumor cells. The aim of this study was to produce the scFv version of pertuzumab. The synthetic scFv gene was cloned into the pET22b expression vector. E. coli BL21 (DE3) and Origami(DE3) were used as hosts. Expression was evaluated in periplasmic and cytoplasmic expression systems. Ni-NTA affinity column was used to purify expressed scFv. Binding ability of scFv was evaluated by flow cytometry analysis and cell-based ELISA. Immunofluoresenct staining and immunocytochemistry were performed to evaluate binding ability of scFv. Effect of scFv on HER dimerization was assessed by tyrosine kinase assay. Results: Molecular weight of scFv was estimated to be approximately 27 kDa, which confirmed by SDS-PAGE and Western blotting. In cytoplasmic expression the most of scFv produced as an insoluble protein which can be highly purified. periplasmic expression compared to cytoplasmic expression resulted in production of more soluble scFv. Biological activity of the purified scFv by its binding to HER2 receptor on surface of tumor cells was confirmed. On the other hand scFv was capable of inhibiting phosphorylation of HER2. Conclusion: In this study, we reported an effective strategy for the expression of the scFv version of pertuzumab specific to domain II of the HER2 receptor in E. coli followed by one-step affinity chromatography for purification. The purified scFv showed a promising bioactivity with a selective binding affinity towards HER2-overexpressing tumor cells. This scFv may be a potential candidate to targeting tumor cell overexpressing HER2 receptor.
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| Descriptor
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Single-Chain Antibodies
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Immunoglobulin Fragments
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Antibodies, Monoclonal
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Escherichia coli
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Abedi, Daryoush, Supervisor
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Mir Mohammad Sadeghi, Hamid, Supervisor
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Jafarian Dehkordi, Abbas, Supervisor
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Chou, Perry, Supervisor
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| Translated Title Supplied by Cataloguer
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ساخت و بیان ژن آنتی بادی مونوکلونال تک زنجیره اختصاصی برای دمین II گیرنده HER2 در اشرشیاکولی و بررسی فعالیت بیولوژیک آن
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