Production of IL-15 and lts Functional Study in Mouse Splenocyte Activation

Han, In Sook; Park, Jong Wook

Abstract

After the synthesis of IL-15 cDNA from the total RNA of mouse spleen, it was inserted into the prokaryotic expression vector, pRseta, and eukaryotic expression vector, pcDNA3.0, respectively. Subsequently, the insertion of gene and open reading frame were confirmed by sequencing of each plasmid, respectively. Using pRseta- IL-15 plasmid, the recombinant IL-15 protein was induced by IPTG under BL21 (DE 3) host cells and recombinant IL-15 was expressed at 14.5 KDa with time. Then, IL- 15 was separated by His-tag affinity chromatography and analyzed by SDS-PAGE to yield soluble IL-15 at 14.5 KDa as monomer and 29.0 KDa as dimer. In order to inspect the function and contribution of IL-15, the in vitro experiment was established using mononuclear cells separated from the mouse spleen. After 48h exposure of PHA to mouse splenocyte and 24h treatment with recombinant IL-15, the effects of cytokine inductions inspected against IL-2, IL-6, IL-10, IL-12, IFN-r, and GM-CSF. The results showed that comparing with the control, IL-6 increased, IL-2, IL-12 and IFN-r increased and similar, and GM-CSF decreased. In addition, the direct injection of pcDNA3.0-IL-15 plasmid into mice gave the similar results to in vitro studies. Namely, IL-6 and IL-12 increased, and IL-2, IFN-r and GM-CSF were similar or decreased. IL-10 was not induced in in vitro and in vivo experiments. These results suggested that the IL-15 induce the splenocyte activation and can be an important factor in proliferation and fuction recovery of weakened T-cell.