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.