رکورد قبلیرکورد بعدی

" Construction, expression and biological activity evaluation of a single- chain Fv antibody frament specific for domain II of HER2 receptor in Eshcherichia coli "


center : Isfahan University of Medical Sciences
Document Type : Latin Dissertation
Language of Document : English
Record Number : 103539
Doc. No : T15332
Call number : ‭QW,525,A313c,2014‬
Main Entry : Akbari, Vajihe
Title & Author : 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
College : Schools, Pharmacy
Date : , 2014
Degree : Pharmaceutical Biotechnology, Ph.D
Page No : XIV, 126p.: ill, diag, tab
Note : This thesis is a research project with project ID 189113
Abstract : 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.
Descriptor : Single-Chain Antibodies
: Immunoglobulin Fragments
: Antibodies, Monoclonal
: Escherichia coli
Added Entry : Abedi, Daryoush, Supervisor
: Mir Mohammad Sadeghi, Hamid, Supervisor
: Jafarian Dehkordi, Abbas, Supervisor
: Chou, Perry, Supervisor
Translated Title Supplied by Cataloguer : ساخت و بیان ژن آنتی بادی مونوکلونال تک زنجیره اختصاصی برای دمین ‭II‬ گیرنده ‭HER2‬ در اشرشیاکولی و بررسی فعالیت بیولوژیک آن
کپی لینک

پیشنهاد خرید
پیوستها
عنوان :
نام فایل :
نوع عام محتوا :
نوع ماده :
فرمت :
سایز :
عرض :
طول :
Construction, expression and biological activity evaluati...
akbarivajiheab.pdf
پایان نامه لاتین
متن
application/pdf
1.69 MB
85
85
موجودی
کتابخانه دانشکده داروسازی
نمایش کامل جزئیات | عدم نمایش جزئیات
جزئیاتمحل نگهداریشماره ثبتشناسه بازیابیجلدوضعيتتاريخ برگشت
دانشکده داروسازی15332موجود‭‬
نظرسنجی