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:: Volume 14, Number 3 (Summer 2017) ::
Sci J Blood Transfus Organ 2017, 14(3): 227-236 Back to browse issues page
Cloning and Expression of Recombinant Human CD40L in HEK293 Cell line
F. Shokoohian, Dr. Z. Sharifi, Dr. M. Mohammadi Pour, Dr. F. Yari *
Abstract:   (15 Views)

Abstract
Background and Objectives
CD40L is a membrane protein and a member of the tumor necrosis factor super family (TNFSF) that plays an important role in transferring cell signaling in innate and adaptive immunity. Since this protein plays its role through clustering the receptors on the target cell surface, it seems that the multimeric form of this molecule can have a stronger effect than the natural trimeric form. So in this study we describe cloning and expression of dodecameric soluble CD40L through self multimerizing protein, surfactant protein D (SP-D), in the HEK293 cell line.
 
Materials and Methods
In this experimental study, the SPD-CD40L was designed in silico and cloned in pcDNA3.1(+) plasmid. HEK293 cells were transfected and used as the expression host cells. The expression of SPD-CD40L was determined at the RNA level by RT-PCR method. After the purification of the recombinant proteins by affinity chromatography, the molecular weight of the recombinant protein was determined by SDS-PAGE. To evaluate the specificity of protein, ELISA and Dot Blot were used.
 
Results
The results indicated that HEK293 cells were transfected and recombinant protein was expressed 24 and 48 hours after Transfection. Moreover, the results of ELISA and Dot Blot methods represented the specificity of recombinant SPD-CD40L chimeric protein.
 
Conclusions 
Chimeric protein SPD-CD40L was expressed successfully by pcDNA3.1(+) in eukaryotic HEK293 cell line and the molecular weight of 4-trimeric (dodecameric) protein was shown to be consistent with the expected amount.
 

 

Keywords: Key words: CD40 Ligand, HEK293 Cells, Transfection
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Type of Study: Research | Subject: Biotechnology
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Shokoohian F, Sharifi Z, Mohammadi Pour M, Yari F. Cloning and Expression of Recombinant Human CD40L in HEK293 Cell line. Sci J Blood Transfus Organ. 2017; 14 (3) :227-236
URL: http://bloodjournal.ir/article-1-1114-en.html
Volume 14, Number 3 (Summer 2017) Back to browse issues page
فصلنامه پژوهشی خون Scientific Journal of Iran Blood Transfus Organ
The Scientific Journal of Iranian Blood Transfusion Organization - Copyright 2006 by IBTO
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