Expression, antibody generation, and biological analysis of chicken interleukin-18.

The gene encoding mature chicken interleukin-18 (ChIL-18) was cloned into prokaryotic expression vector pET-30a(+), resulting in a recombinant plasmid pET-30a-ChIL-18. After pET-30a-ChIL-18 was transformed into Escherichia coli Rosseta, the expression of ChIL-18 induced by 1 mM IPTG at 37°C was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The expressed fusion protein of 26 kDa was purified with a Ni-NTA affinity column and used to generate a hyperimmune antiserum in a rabbit. The specificity and titer of anti-ChIL-18 serum were analyzed by the enzyme-linked immunosorbent assay. Western blot and immunofluorescence assays indicated that the anti-ChIL-18 antibody specifically reacted with the ChIL-18 expressed from E. coli or ChIL-18-transfected eukaryotic cells. Moreover, the renatured ChIL-18 stimulated the production of nitric oxide (NO) from macrophages via eliciting the secreting of IFN-γ from lymphocytes.