Pouriayevali, Mohammah Hassan, Memarnejadian, Arash Reza, Sadat, Seyad Mehdi, Zavva, Mahdi, Siadat, Seyad Davar, Hartoonian, Christine, Aghasadeghi, Mohammad Reza (2011). Designing and construction of bicistronic plasmid pIRES-Igk/mIL18/Fc: potential implications for vaccine investigations. , 14(2), 13-23.
Mohammah Hassan Pouriayevali; Arash Reza Memarnejadian; Seyad Mehdi Sadat; Mahdi Zavva; Seyad Davar Siadat; Christine Hartoonian; Mohammad Reza Aghasadeghi. "Designing and construction of bicistronic plasmid pIRES-Igk/mIL18/Fc: potential implications for vaccine investigations". , 14, 2, 2011, 13-23.
Pouriayevali, Mohammah Hassan, Memarnejadian, Arash Reza, Sadat, Seyad Mehdi, Zavva, Mahdi, Siadat, Seyad Davar, Hartoonian, Christine, Aghasadeghi, Mohammad Reza (2011). 'Designing and construction of bicistronic plasmid pIRES-Igk/mIL18/Fc: potential implications for vaccine investigations', , 14(2), pp. 13-23.
Pouriayevali, Mohammah Hassan, Memarnejadian, Arash Reza, Sadat, Seyad Mehdi, Zavva, Mahdi, Siadat, Seyad Davar, Hartoonian, Christine, Aghasadeghi, Mohammad Reza Designing and construction of bicistronic plasmid pIRES-Igk/mIL18/Fc: potential implications for vaccine investigations. , 2011; 14(2): 13-23.

Designing and construction of bicistronic plasmid pIRES-Igk/mIL18/Fc: potential implications for vaccine investigations

Pathobiology Reserach
Article 2, Volume 14, Issue 2, 2011, Pages 13-23    PDF (732.49 K)
Authors
Mohammah Hassan Pouriayevali1; Arash Reza Memarnejadian2; Seyad Mehdi Sadat3; Mahdi Zavva4; Seyad Davar Siadat5; Christine Hartoonian6; Mohammad Reza Aghasadeghi* 2
1Department of Hepatitis and AIDS, Pasteur Institute of Iran, Tehran
2Asisstant Professor, Department of Hepatitis and AIDS, Pasteur Institute of Iran
3Ph.D., Department of Hepatitis and AIDS, Pasteur Institute of Iran,
4Department of Hepatitis and AIDS, Pasteur Institute of Iran
5Associated Professor, Department of Hepatitis and AIDS, Pasteur Institute of Iran
6Ph.D, Department of Virology, Pasteur Institute of Iran
Abstract
Objective: IL18 is a cytokine that plays an important role in the T-cell-helper type 1 (Th1) response and hence, plasmid-encoded IL18 is considered as a potent genetic adjuvant for DNA vaccine studies. In this study, a bicistronic eukaryotic plasmid capable of secreting a more stable mouse IL18 (fused with Fcγ2a fragment) was constructed and expression of this chimer cytokine was also assessed.
Materials and Methods: RNA purified from stimulated mouse spleenocytes and then cDNA corresponding to mouse IL18 (mIL18) and Fcγ2a fragments were constructed by RT-PCR. Sequential subcloning of mIL18 and IgG2aFc fragments first into pSL1180 and then pSecTag2 plasmids resulted in the fusion of mIL18/Fc and addition of immunoglobulin kappa signal sequence (Igk/mIL18/Fc), respectively. Final cloning of Igk/mIL18/Fc sequence downstream of CMV promoter into the NheI/XmaI sites of pIRES2-GFP plasmid and led to the construction of pIRES-Igk/mIL18/Fc plasmid, which was transfected to HEK293T cell line by Turbofec Transfection reagent and expression analysis, was evaluated by ELISA assay.
Results: Restriction enzyme analysis of pSL-mIL18، pSL-mIL18/Fc، pSec-mIL18/Fc and pIRES- Igk/mIL18/Fc plasmids with the enzymes that were applied for clonings led to the isolation of fragments with expected size and then plasmid of pIRES- Igk/mIL18/Fc was also confirmed following sequencing reactions. Moreover, expression and secretion of mIL18 to the medium was evidenced in transfected 293T cells, compared to non-transfected controls.
Conclusion: pIRES- Igk/mIL18/Fc plasmid possesses the capacity of the cloning and expression of putative antigen gene under the direction of IRES sequence, and also expression of mIL18 as a great secretive genetic adjuvant. This results can be useful to design an efficient DNA vaccine especially for inducing host cellular immune response, moreover, cab be considered a promising for accessing to new generation of DNA vaccine.
Keywords
IRES; Bicistronic Plasmid; Genetic Adjuvant; IL-18
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