Volume 21, Number 2 (Scientific Journal of Hamadan University of Medical Sciences-Summer 2014)                   Sci J Hamadan Univ Med Sci 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. Sci J Hamadan Univ Med Sci . 2014; 21 (2) :145-151
URL: http://sjh.umsha.ac.ir/article-1-94-en.html

Associate Professor of Biotechnology, Molecular Medicine Research Center, Hamadan University of Medical Sciences & Health Services, Hamadan, Iran. , sjam110@yahoo.com
Abstract:   (2322 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]   (854 Downloads)    
Type of Study: Original | Subject: Special

Add your comments about this article : Your username or email:
Write the security code in the box

Send email to the article author


© 2015 All Rights Reserved | Scientific Journal of Hamadan University of Medical Sciences

Designed & Developed by : Yektaweb