Designing and construction of bicistronic plasmid pIRES-Igk/mIL۱۸/Fc: potential implications for vaccine investigations

Objective: IL۱۸ is a cytokine that plays an important role in the T-cell-helper type ۱ (Th۱) response and hence, plasmid-encoded IL۱۸ 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 IL۱۸ (fused with Fcγ۲a 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 IL۱۸ (mIL۱۸) and Fcγ۲a fragments were constructed by RT-PCR. Sequential subcloning of mIL۱۸ and IgG۲aFc fragments first into pSL۱۱۸۰ and then pSecTag۲ plasmids resulted in the fusion of mIL۱۸/Fc and addition of immunoglobulin kappa signal sequence (Igk/mIL۱۸/Fc), respectively. Final cloning of Igk/mIL۱۸/Fc sequence downstream of CMV promoter into the NheI/XmaI sites of pIRES۲-GFP plasmid and led to the construction of pIRES-Igk/mIL۱۸/Fc plasmid, which was transfected to HEK۲۹۳T cell line by Turbofec Transfection reagent and expression analysis, was evaluated by ELISA assay. Results: Restriction enzyme analysis of pSL-mIL۱۸، pSL-mIL۱۸/Fc، pSec-mIL۱۸/Fc and pIRES- Igk/mIL۱۸/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/mIL۱۸/Fc was also confirmed following sequencing reactions. Moreover, expression and secretion of mIL۱۸ to the medium was evidenced in transfected ۲۹۳T cells, compared to non-transfected controls. Conclusion: pIRES- Igk/mIL۱۸/Fc plasmid possesses the capacity of the cloning and expression of putative antigen gene under the direction of IRES sequence, and also expression of mIL۱۸ 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.

کلیدواژه ها:

IRES ، IRES ، Bicistronic Plasmid ، Genetic Adjuvant ، IL-۱۸ ، پلاسمید جفت بیانی ، ادجوانت ژنی ، اینترلوکین ۱۸