Cloning, Expression And Relatively Functional Characterization Of IL-12p40in Oplegnathus Fasciatus

IL-12p40, also called IL-12β, is a subunit of the proinflammatory cytokines interleukin(IL)-12and IL-23，which is produced predominantly by activated monocytes, macrophages, anddendritic cells. In mammals， IL-12p40is encoded by the IL12p40gene located on5q31–33and has pro-infammatory and immunoregulatory activities. In teleost, three IL-12p40isoforms,p40a, p40b, p40c, have been identified in several species, including sea bass (Dicentrarchuslabrax L.), Atlantic halibut (Hippoglossus hippoglossus L.), common carp (Cyprinus), however,the biological function of fish IL-12p40is essentially unknown.In this work, we reported the identification and gene structure and biological analysis of anIL-12p40, OfIL-12p40, from rock bream (Oplegnathus fasciatus).The cDNA of OfIL-12p40contains a5’–UTR of81bp, an ORF of1086bp, and a3’–UTRof642bp. The3’–UTR has two ATTTA motifs that are typically found in cytokine mRNA.OfIL-12p40is composed of361amino acids and possesses a conserved IL-12p40domain and aWSxWS signature motif characteristic of known IL-12p40. Five cysteine residues, i.e. C44, C92,C120, C131, and C156, of OfIL-12p40are completely conserved in fish, human, and mouseIL-12p40, while three cysteine residues, i.e. C127, C149, and C223, of OfIL-12p40are present onlyin fish IL-12p40. Sequence alignment showed that OfIL-12p40shares46-57%overall sequenceidentities with the IL-12p40homologues of many teleost species. Phylogenetic analysisindicated that OfIL-12p40belongs to p40c.The result of qRT-PCR analysis showed that constitutive expression of OfIL-12p40occurred in multiple tissues including brain, gill, heart, liver, spleen, muscle, intestine, kidney,and was lowest in brain, highest in kidney. Experimental infection with bacterial pathogenupregulated the expression of OfIL-12p40in kidney and spleen, with peak inductions occurringat48h (16.3-fold) and24h post-infection (6.9-fold) respectively. In order to find out thebiological function of OfIL-12p40, we expressed OfIL-12p40in vitro and purified recombinant OfIL-12p40(rOfIL-12p40). Biological function analysis showed that rOfIL-12p40stimulatedthe respiratory burst activity of peripheral blood leukocytes in a dose-dependent manner.rOfIL-12p40also enhanced the resistance of rock bream against bacterial infection andupregulated the expression of innate immune genes in kidney, including IL-1β，IFN1，CCK1，IRF-4，ISG15å'ŒC7（18.6,7.5,14.9,8.0,6.8and24.6-fold respectively）. Taken together, theseresults indicate that OfIL-12p40possesses cytokine-like property and plays a role in immunedefense against bacterial infection.