Neospora caninum: cloning and expression of a gene coding for cytokine-inducing profilin

Profilins are actin-binding proteins that in Toxoplasma gondii stimulate innate immunity in mice by binding Toll-like receptors (TLR) on dendritic cells (DC) leading to release of inflammatory cytokines, primarily IL-12 and IFN-gamma. The purpose of the present study was to characterize Neospora caninum profilin, termed NcProfilin. Recombinant NcProfilin was purified by affinity chromatography, and used to prepare specific antisera to allow characterization of native NcProfilin antigen in N. caninum tachyzoites. By immunoblotting, recombinant NcProfilin is 22kDa, and is similar in size to the respective 22kDa native protein. Immunofluorescence and immunoelectron microscopy localized native NcProfilin to the apical end of N. caninum tachyzoites. Incubation of recombinant NcProfilin with spleen cells from BALB/c mice induced release of IFN-gamma. Also, injection of BALB/c mice with purified rNcProfilin elicited a strong IFN-gamma and IL-12 responses at 6 and 24h after injection indicating that NcProfilin may be an important protein in regulation of cytokine responses to N. caninum.