Cloning and characterization of tumor necrosis factor superfamily 15 in rock bream, Oplegnathus fasciatus; phylogenetic, in silico, and expressional analysis.

Tumor necrosis factor superfamily (TNFSF)15 is a member of TNFSF which shares a high homology with other TNFSFs, especially lymphotoxin (LT)-α in teleost. In this study, we have cloned a putative TNFSF15 gene in rock bream which was highly homologous with other fish TNFSF15 and performed bioinformatic analysis to confirm the membership. The RB-TNFSF15 cDNA consists of 3192 bp (193 bp of 5'-untranslated region (UTR), 732 bp of ORF, and 2267 bp of 3'-UTR) and encodes a polypeptide of 243 amino acids containing a predicted TNF superfamily signature with 43-61% identities with fish TNFSF15. The predicted 3D structure was similar to human TNFSF15 with β barrel structure containing 10 β strands and 1 α helix while human LT-α and β contain 10 β strands and 2 α helices. Consequently, the synteny and phylogenetic analysis of fish TNFSF15 genes and structural similarity of the predicted protein to mammalian TNFSF15 implicate that they can be identified as TNFSF15. In healthy rock bream, RB-TNFSF15 gene expression level was the highest in fin and the lowest in blood. In vitro, TNFSF15 gene expression was up-regulated by lipopolysaccharide, polyinosinic:polycytidylic acid (poly I:C) and rock bream iridovirus (RBIV) in head kidney, while up-regulated by poly I:C and RBIV at later time in spleen. In vivo, RB-TNFSF15 gene expression was up-regulated in head kidney, liver and blood after vaccination with a formalin inactivated RBIV. After challenging with RBIV, RB-TNFSF15 gene expression was up-regulated in unvaccinated group at day 3 post-infection in head kidney. In gill, it was significantly up-regulated in vaccinated group at day 1 post-challenge and all groups at day 7, indicating that RB-TNFSF may play a key role in mucosal immunity during viral infection. Since the regulation mechanism of TNFSF15 gene expression in fish has not yet been elucidated, the present study will help to understand the roles of TNFSF15 in fish immune system.