Nitric Oxide Inducing Function and Intracellular Movement of Chicken Interleukin‐18 in Cultured Cells

To evaluate the characteristics of chicken interleukin‐18 (ChIL‐18) in different forms in vitro, the ChIL‐18 full‐length gene (ChIL‐18‐F) and the ChIL‐18 presumed mature protein gene (ChIL‐18‐M) were cloned and inserted into the eukaryotic expression vector pCI, to construct recombinant pCI‐ChIL‐18‐F and pCI‐ChIL‐18‐M. The recombinant plasmids were then transferred into chicken splenic lymphocytes (CSLs). Western blot showed that ChIL‐18‐F, with a molecular weight of 23.0 kDa, was produced in CSLs transfected by pCI‐ChIL‐18‐F; ChIL‐18‐M, with a molecular weight of 19.5 kDa, was produced in CSLs transfected by pCI‐ChIL‐18‐M. The nitric oxide (NO) level in the transfected CSLs and the culture medium at different time points was further examined under confocal microscopy using 4,5‐diaminofluorescein staining. The results showed that both pCI‐ChIL‐18‐F and pCI‐ChIL‐18‐M groups showed significant increase in intracellular and extracellular NO production compared with pCI transfected control cells. These results suggest that both ChIL‐18‐F and ChIL‐18‐M could stimulate NO secretion in CSLs. To characterize the intracellular distribution of ChIL‐18, ChIL‐18‐F and ChIL‐18‐M were each fused to the enhanced green fluorescent protein gene, and expressed in Vero cells. The results showed that the ChIL‐18‐F tended to the membranous region in Vero cells, while ChIL‐18‐M did not. This indicates that the N‐terminal 27 amino acid peptide helped ChIL‐18 target to Vero cell membranes. Edited by Chang‐De LU