DESIGNING AND CONSTRUCTION OF BICISTRONIC PLASMID PIRES-IGK/MIL18/FC: POTENTIAL IMPLICATIONS FOR VACCINE INVESTIGATIONS

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POURIAYEVALI MOHAMMAH HASSAN; MEMARNEJADIAN ARASH REZA; SADAT SEYAD MEHDI; ZAVVA MAHDI; SIADAT SEYAD DAVAR; HARTOONIAN CHRISTINE; AGHASADEGHI MOHAMMAD REZA

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Journal: Pathobiology Research Year:2011 | Volume:14 | Issue:2 Page(s): 13-23

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Title

DESIGNING AND CONSTRUCTION OF BICISTRONIC PLASMID PIRES-IGK/MIL18/FC: POTENTIAL IMPLICATIONS FOR VACCINE INVESTIGATIONS

Author(s)

Keywords

IL-18Q2

BICISTRONIC PLASMIDQ2

GENETIC ADJUVANTQ2

IRESQ2

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 Fcg2a 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 Fcg2a 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.

Cites

DESIGN, CLONING, AND EXPRESSION ASSAY OF THE NP GENE IN A BICISTRONIC VECTOR HARBORING THE MOUSE IL-18 GENE: POTENTIAL IMPLICATIONS FOR TYPE A INFLUENZA VACCINE INVESTIGATIONS

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APA: Copy

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. PATHOBIOLOGY RESEARCH (MODARES JOURNAL OF MEDICAL SCIENCES), 14(2), 13-23. SID. https://sid.ir/paper/81318/en

Vancouver: Copy

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. PATHOBIOLOGY RESEARCH (MODARES JOURNAL OF MEDICAL SCIENCES)[Internet]. 2011;14(2):13-23. Available from: https://sid.ir/paper/81318/en

IEEE: Copy

MOHAMMAH HASSAN POURIAYEVALI, ARASH REZA MEMARNEJADIAN, SEYAD MEHDI SADAT, MAHDI ZAVVA, SEYAD DAVAR SIADAT, CHRISTINE HARTOONIAN, and MOHAMMAD REZA AGHASADEGHI, “DESIGNING AND CONSTRUCTION OF BICISTRONIC PLASMID PIRES-IGK/MIL18/FC: POTENTIAL IMPLICATIONS FOR VACCINE INVESTIGATIONS,” PATHOBIOLOGY RESEARCH (MODARES JOURNAL OF MEDICAL SCIENCES), vol. 14, no. 2, pp. 13–23, 2011, [Online]. Available: https://sid.ir/paper/81318/en

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