1. Tarbiat Modares University 2. Tarbiat Modares University 3. Tehran University of Medical Sciences (TUMS) 4. National Institute of Genetic Engineering and Biotechnology (NIGEB)
Abstract
Introduction
Due to texture and small size, identifying the parathyroid from thyroid glands is a very difficult procedure. they should be preserved during thyroidectomy to avoid improper side effects of hypocalcemia. it has been demonstrated that antibodies can be used in vivo as diagnosis tools in non-invasive imaging. it seems that a labeled antibody against one of the highly expressed receptors on the surface of parathyroid glands can be revealed an appropriate candidate to distinguish them from surrounding tissues. therefore, a membrane of g-protein-coupled receptors (gpcrs) named as calcium sensing receptor (casr) was selected.
Methods
First at all, we intend to select the best antigenic regions of the casr using various bioinformatics tools. then, the best immunological region was selected and consequently the corresponding gene was cloned into the pet28a expression vector in e. coli bl21 (de3). to optimize protein expression, we examined different concentrations of iptg (1, 0.5 and 0.1 mm) at several temperatures including 16, 28 and 37c for 18, 12 and 4 h, respectively.
Results
However, protein was expressed as inclusion bodies. following, recombinant protein was purified using ni-nta resin via denaturing method and verified on 12.5% sds. step wise dialysis was done to remove urea and refold protein.
Conclusion
The results demonstrated that truncated form of casr antigen can be expressed and purified, properly. we hope this purified protein can be used to product monoclonal antibodies and found an effective detection system.
