Volume 21, Issue 2 (Scientific Journal of Hamadan University of Medical Sciences-Summer 2014)                   Avicenna J Clin Med 2014, 21(2): 145-151 | Back to browse issues page

XML Persian Abstract Print


Download citation:
BibTeX | RIS | EndNote | Medlars | ProCite | Reference Manager | RefWorks
Send citation to:

Barati G, Mirza Hosseini H, Karimi J, Ebrahimzadeh F, Shabab N, Saidijam M. Human Interleukine-1 receptor antagonist:Cloning, Expression and Optimization in E.coli Host. Avicenna J Clin Med 2014; 21 (2) :145-151
URL: http://sjh.umsha.ac.ir/article-1-94-en.html
1- , sjam110@yahoo.com
Abstract:   (9089 Views)

Introduction & Objective: Interleukine-1 receptor antagonist (IL-1RA) is a powerful anti-inflammatory cytokine which limits the biological effects of IL-1. Due to structural similarity between IL-1 and its antagonist, IL-1RA competitively binds to IL-1 receptor which leads to no signal transduction. Therefore , it is applied in the treatment of patients with inflammatory diseases such as Rheumatoid Arthritis. The aim of this study is cloning, expression and op-timization of IL-1RA in E. coli.

Materials & Methods: In this experimental study synthetically prepared cDNA was amplified by PCR. After double digestion with NdeI and XhoI restriction enzymes, this gene was cloned in pET28a expression vector. Expression of desired gene was analyzed at RNA level by RT-PCR and at protein level by SDS-PAGE and followed by western blot to confirm SDS-PAGE results. Optimization of recombinant protein expression was performed in dif-ferent IPTG concentrations and harvesting times after induction.

Results: The presence of gene in pET28a was determined by colony-PCR and confirmed by restriction digestion. Transcription of cloned gene and expression of high yield recombinant protein were shown by RT-PCR and SDS-PAGE, respectively. The result of SDS-PAGE was confirmed by western blot. Expression was optimized in different induction time and IPTG concentrations

Conclusion: The result of this study demonstrated expression of this recombinant protein at high level in E.coli system by pET28a expression vector. This study also showed a direct as-sociation between the increased level of expression and time of induction . Therefore, an overnight induction time with 0.1 mM IPTG concentration is recommended for a high level expression.

Full-Text [PDF 277 kb]   (6967 Downloads)    
Type of Study: Original | Subject: Other Clinical Specialties

Add your comments about this article : Your username or Email:
CAPTCHA

Send email to the article author


Rights and permissions
Creative Commons License This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.

© 2024 CC BY-NC 4.0 | Avicenna Journal of Clinical Medicine

Designed & Developed by : Yektaweb