Intein-mediated expression and purification of common carp IFN-gamma and its protective effect against spring viremia of carp virus

摘要

IFN-gamma is a pleiotropic cytokine with significant roles in antiviral, antitumor and immune regulation. It could be used as an immuno-enhancer to improve fish protectiveness against pathogens. In this study, the prokaryotic expression plasmid pTwin1-N-IFN-gamma was constructed to express Cyprinus carpio (common carp) IFN-gamma fused with a chitin binding domain (CBD) and a self-cleavable intein-tag, Synechocystis sp DnaB. The recombinant protein CBD-DnaB-IFN-gamma with the molecular weight of 44.25 kD was successfully expressed in soluble form, and the rIFN-gamma (approximate 18.61 kD) was further cleaved and eluted under pH = 7.0 at 25 degrees C. rIFN-gamma could be recognized by western blotting with rabbit anti-grass carp IFN-gamma polydonal antibody. Cytotoxicity studies on EPC cells showed that only 500 ng/ml rIFN-gamma had a subtle effect on cells growth and its proliferation rate was reduced to 76.2%. EPC cells incubated with 100 ng/ml rIFN-gamma showed significantly higher resistance against SVCV, reducing the TCID50/ml by more than 800-fold. In vivo studies suggested that intraperitoneal injection of rIFN-gamma significantly improved the survival rate of common carps compared with SVCV challenge alone. These results implied that rIFN-gamma would act as an immuno-enhancer in carp aquaculture.