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

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.