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کاربرد نوع شرط:
- جایگاه : پژوهشی
- مجله: Iranian Journal of Biotechnology
- نوع مقاله: Journal Article
- کلمات کلیدی: Purification,Protein expression,Polyclonal antibody,FCRL,His-tag
- چکیده:
- چکیده انگلیسی: Background: The Fc receptor like (FCRL) molecules belong to the immunoglobulin (Ig) superfamily with potentially immunoregulatory function. Among the FCRL family FCRL2 and 4 are predominantly expressed on memory B cells and FCRL1 is a pan- B cell marker. To date, no ligand has been identifid for the human FCRL1, 2 and 4 molecules. Objectives: Cloning, expression, purifiation and structural analysis of the extracellular domain of human FCRL1, 2 and 4 proteins. Materials and Methods: In this study, the extracellular part of human FCRL1, 2 and 4 were subcloned into prokaryotic expression vectors pET-28b (+) and transformed into BL21-DE3 E.coli strain. Protein expression was optimized by fie adjustments such as induction time, incubation temperature and expression hosts. Recombinant FCRL proteins were purifid by metal affity chromatography using Ni-NTA resin. Purifid FCRL proteins were further characterized by SDS-PAGE and immunoblotting using His-tag and FCRL specifi polyclonal antibodies. Results: Our results demonstrated that FCRL1, 2 and 4 were successfully expressed in pET-28b (+) vector. Optimization of the expression procedure showed that IPTG induction at OD600 = 0.9 and overnight incubation at 37˚C resulted in the highest expression levels of FCRL proteins ranging from approximately 15% (FCRL1) to 25% (FCRL2 and 4) of the total bacterial lysate proteins. Conclusions: These purifid recombinant proteins are potentially a valuable tool for investigating the immunoregulatory function of FCRL molecules and the production of specifi mAbs for immunotherapeutic interventions.
- انتشار مقاله: 04-10-1391
- نویسندگان: Mahdi Shabani,Azam Hemmati,Mahdi Zandemami,Jalal Khoshnoodi,Mahmood Jeddi-Tehrani,Hodjatallah Rabbani,Zahra Amirghofran,Fazel Shokri
- مشاهده
- جایگاه : پژوهشی
- مجله: Asian Pacific Journal of Cancer Prevention
- نوع مقاله: Journal Article
- کلمات کلیدی: Breast cancer,Fusion protein,HER3,Polyclonal antibody
- چکیده:
- چکیده انگلیسی: Objective: Human epidermal growth factor receptor 3 (HER3) is a unique member of the tyrosine kinase receptors with an inactive kinase domain and is the preferable dimerization partner for HER2 which lead to potent tumorigenic signaling. Methods: In this study, the expression plasmids coding for the human HER3 subdomains were transfected into CHO-K1 cells. Produced proteins were characterized by ELISA and SDS-PAGE. Rabbits were immunized and produced polyclonal antibodies (pAbs) that were characterized by ELISA, Immunoblotting and flowcytometry and their inhibitory effects were assessed by XTT on BT-474 and JIMT-1 breast cancer cell lines. Result: The recombinant subdomains were highly immunogenic in rabbits. The pAbs reacted with the recombinant subdomains as well as commercial HER3 and the native receptor on tumor cell membranes and could significantly inhibit growth of Trastuzumab sensitive (BT-474) and resistant (JIMT-1) breast cancer cell lines in vitro. Conclusion: It seems that HER3 extra cellular domains (ECD) induce a strong anti-tumor antibody response and may prove to be potentially useful for immunotherapeutic applications.
- انتشار مقاله: 21-05-1398
- نویسندگان: Samaneh Mansouri-Fard,Mojgan Ghaedi,Mohammad-Reza Shokri,Tannaz Bahadori,Jalal Khoshnoodi,Forough Golsaz-Shirazi,Mahmood Jeddi-Tehrani,Mohammad Mehdi Amiri,Fazel Shokri
- مشاهده