|
Cloning and Expression of Recombinant Human CD40L in HEK293 Cell line
|
F. Shokoohian   , Z. Sharifi , M. Mohammadi Pour , F. Yari |
|
|
| Keywords: Key words: CD40 Ligand, HEK293 Cells, Transfection |
|
|
Full-Text [PDF 621 kb]
(1930 Downloads)
| Abstract (HTML) (4778 Views)
|
Type of Study: Research |
Subject:
Biotechnology
Published: 2017/09/12
|
|
|
|
|
|
|
Full-Text: (2527 Views) |
References:
- Kornbluth RS. The emerging role of CD40 ligand in HIV infection. J Leukoc Biol 2000; 68(3): 373-82.
- Kim HO, Kim HS, Youn JC, Shin EC, Park S. Serum cytokine profiles in healthy young and elderly population assessed using multiplexed bead-based immunoassays. J Transl Med 2011; 9(1): 113.
- Galicia López A, Olguín Ortega L, Saavedra MA, Méndez Cruz R, Jimenez Flores R, García de la Peña M.. Increased concentrations of soluble CD40 ligand platelet in patients with primary antiphospholipidic syndrome. Reumatol Clín 2013; 9(4): 216-20. [Article in English, Spanish]
- Aloui C, Prigent A, Sut C, Tariket S, Hamzeh-Cognasse H, Pozzetto B, et al. The signaling role of CD40 ligand in platelet biology and in platelet
component transfusion. Int J Mol Sci 2014; 15(12): 22342-64 .
- Danese S, Sans M, Fiocchi C. The CD40/CD40L costimulatory pathway in inflammatory bowel disease. Gut 2004; 53(7): 1035-43.
- Davidson DC, Jackson JW, Maggirwar SB. Targeting platelet-derived soluble CD40 ligand: a new treatment strategy for HIV-associated neuroinflammation? J Neuroinflammation 2013; 10: 144.
- Elgueta R, Benson MJ, De Vries VC, Wasiuk A, Guo Y, Noelle RJ. Molecular mechanism and function of CD40/CD40L engagement in the immune system. Immunol Rev 2009; 229(1): 152-72.
- Antoniades C, Bakogiannis C, Tousoulis D, Antonopoulos AS, Stefanadis C. The CD40/CD40 ligand system: linking inflammation with atherothrombosis. J Am Coll Cardiol 2009; 54(8): 669-77.
- Daoussis D, Andonopoulos AP, Liossis SN. Targeting
CD40L: a promising therapeutic approach. Clin Diagn Lab Immunol 2004; 11(4): 635-41 .
- Giunta B, Rezai-Zadeh K, Tan J. Impact of the CD40-CD40L dyad in Alzheimer's disease. CNS Neurol Disord Drug Targets 2010; 9(2): 149-55.
- Liu A, Guardino A, Chinsangaram L, Goldstein MJ, Panicali D, Levy R. Therapeutic vaccination against murine lymphoma by intratumoral injection of recombinant fowlpox virus encoding CD40 ligand. Cancer Res 2007; 67(14): 7037-44.
- Aversa G, Punnonen J, Carballido JM, Cocks BG, de Vries JE. CD40 ligand-CD40 interaction in Ig isotype switching in mature and immature human B cells. Semin Immunol 1994; 6(5): 295-301.
- Blumberg N, Spinelli SL, Francis CW, Taubman MB, Phipps RP. The platelet as an immune cell-CD40 ligand and transfusion immunomodulation. Immunol Res 2009; 45(2-3): 251-60.
- Cognasse F, Boussoulade F, Chavarin P, Acquart S, Fabrigli P, Lamy B, et al. Release of potential immunomodulatory factors during platelet storage. Transfusion 2006; 46(7): 1184-9.
- Esmaili MA, Yari F, Sharifi Z, Nikougoftar M, Fadaei R. Effects of platelet microparticles on the activation of B cells. Modares Journal of Medical Sciences: Pathobiology 2013; 15(4): 1-10. [Article in Farsi]
- Ma DY, Clark EA. The role of CD40 and CD154/CD40L in dendritic cells. Semin Immunol 2009; 21(5): 265-72.
- Spriggs M, Armitage R, Strockbine L, Clifford K, Macduff B, Sato T, et al. Recombinant human CD40 ligand stimulates B cell proliferation and immunoglobulin E secretion. J Exp Med 1992; 176(6): 1543-50.
- Garcia-Marquez MA, Shimabukuro-Vornhagen A, Theurich S, Kochanek M, Weber T, Wennhold K, et al. A multimerized form of recombinant human CD40 ligand supports long-term activation and proliferation of B cells. Cytotherapy 2014; 16(11): 1537-44.
- Haswell LE, Glennie MJ, Al-Shamkhani A. Analysis of the oligomeric requirement for signaling by CD40 using soluble multimeric forms of its ligand, CD154. Eur J Immunol 2001; 31(10): 3094-100.
- Kilinc MO, Mukundan L, Yolcu ES, Singh NP, Suttles J, Shirwan H. Generation of a multimeric form of CD40L with potent immunostimulatory activity using streptavidin as a chaperon. Exp Mol Pathol 2006; 80(3): 252-61.
- Song W, Levy R. Therapeutic vaccination against murine lymphoma by intratumoral injection of naive dendritic cells. Cancer Res 2005; 65(13): 5958-64.
- Contin C, Pitard V, Itai T, Nagata S, Moreau JF, Déchanet-Merville J. Membrane-anchored CD40 Is Processed by the Tumor Necrosis Factor-alpha-converting enzyme. Implications for CD40 signaling. J Biol Chem 2003; 278(35): 32801-9.
- Croft M, Benedict CA, Ware CF. Clinical targeting of the TNF and TNFR superfamilies. Nat Rev Drug Discov 2013; 12(2): 147-68.
- Kornbluth RS. Nucleic Acids Encoding Multimeric Fusion Proteins of TNF Superfamily Ligands [US20090263348 A1]. Google Patents; 2009.
- Schuchmann M, Hess S, Bufler P, Brakebusch C, Wallach D, Porter A, et al. Functional discrepancies between tumor necrosis factor and lymphotoxin α explained by trimer stability and distinct receptor interactions. Eur J Immunol 1995; 25(8): 2183-9.
- Wright JR. Immunoregulatory functions of surfactant proteins. Nat Rev Immunol 2005; 5(1): 58-68.
- Chen C. Challenges and opportunities of monoclonal antibody manufacturing in China. Trends in Bio/Pharmaceutical Industry 2009; 5(3): 28-33.
- Holler N, Tardivel A, Kovacsovics-Bankowski M, Hertig S, Gaide O, Martinon F, et al. Two adjacent trimeric Fas ligands are required for Fas signaling and formation of a death-inducing signaling complex. Mol Cell Biol 2003; 23(4): 1428-40.
- Jiang YF, He Y, Gong GZ, Chen J, Yang CY, Xu Y. Construction of recombinant eukaryotic expression plasmid containing murine CD40 ligand gene and its expression in H22 cells. World J Gastroenterol 2005; 11(2): 182-6.
- Stone GW, Barzee S, Snarsky V, Kee K, Spina CA, Yu X-F, et al. Multimeric soluble CD40 ligand and GITR ligand as adjuvants for human immunodeficiency virus DNA vaccines. J Virol 2006; 80(4): 1762-72.
- Kornbluth RS. Nucleic acids encoding multimeric proteins of TNF superfamily ligands [US7332298 B2]. Google Patents; 2008.
|
Sci J Iran Blood Transfus Organ 2017; 14(3): 227-236
|
Cloning and Expression of Recombinant Human
CD40L in HEK293 Cell line
Shokoohian B.1, Sharifi Z.1, Mohammadi Pour M.1, Yari F.1
1Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran
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.
Key words: CD40 Ligand, HEK293 Cells, Transfection
Received: 15 Apr 2017
Accepted: 13 Jun 2017
Correspondence: Yari F., PhD of Immunology. Associate Professor of Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine.
P.O.Box: 14665-1157, Tehran, Iran. Tel: (+9821) 82052237; Fax: (+9821) 88601555
E-mail: f.yari@ibto.ir
|
|
| Send email to the article author |
|
|
|
| Add your comments about this article |
|
|
|
|
Shokoohian F, Sharifi Z, Mohammadi Pour M, Yari F. Cloning and Expression of Recombinant Human CD40L in HEK293 Cell line. Sci J Iran Blood Transfus Organ 2017; 14 (3) :227-236 URL: http://bloodjournal.ir/article-1-1114-en.html
|
.jpg)
.png)
