Function Study Of Two Immune-related Genes Of Cynoglossus Semilaevis, HMGB2 And IFIT1

In this study, we have identified two immune related genes HMGB2(high-mobility group protein B2) and IFIT1(Interferon-induced protein with tetratricopeptide repeats1) from half smooth tongue sole(Cynoglossus semilaevis), and analyzed their functions in host defense pathogen. We also determinated the best internal references for qRT-PCR analysis of gene expression while the fish was infected with pathogen.High mobility group box(HMGB) proteins are known to be involved in diverse functions in mammalian cells. In teleost, very limited studies on HMGB proteins have been documented. In this study, we reported identification of a HMGB homologue(named CsHMGB2) from tongue sole and examined its biological property. CsHMGB2 is 245 residues in length and contains two basic HMG boxes and an acidic C-terminal tail composed of 23 Asp/Glu residues. Quantitative real time RT-PCR(qRT-PCR) analysis showed that CsHMGB2 expression occurred in multiple tissues and was upregulated by bacterial and viral infection in a time-dependent manner. In vitro studies showed that when tongue sole peripheral blood leukocytes were treated with recombinant CsHMGB2(rCsHMGB2) and the mutant rCsHMGB2 M, which bears a deletion of the C-terminal acidic region, significant and comparable increases in cellular resistance against bacterial infection were observed. qRT-PCR detected enhanced expression of proinflammatory cytokines and chemokines in rCsHMGB2-treated cells. In vivo studies showed that when tongues sole were administered with rCsHMGB2 or r CsHMGB2 M before being subjected to bacterial and viral infection, the pathogen loads in the spleen and kidney of the fish were significantly reduced. Taken together, these results suggest that CsHMGB2 possesses immunoregulatory properties that promote resistance against bacterial and viral infection in a manner that is largely independent on the highly conserved C-terminal acidic domain.Interferon-induced protein with tetratricopeptide repeats(IFIT) is involved in antiviral immunity in mammalian cells. In fish, the function of this group of proteins is unknown. In this study, we report the identification of an IFIT homologue(named Cs IFIT1) from tongue sole(Cynoglossus semilaevis) and examined its antiviral potential. CsIFIT1 is composed of 436 residues and shares 30.9%-58.7% overall sequence identities with the known IFITs of a number of teleost species. In silico analysis identified two tetratricopeptide repeats in CsIFIT1. Quantitative real time RT-PCR analysis showed that Cs IFIT1 expression was most abundant in immune relevant organs and upregulated, in a time-dependent manner, by experimental infection with the fish viral pathogen megalocytivirus. When Cs IFIT1 was overexpressed in tongue sole before megalocytivirus infection, viral replication in fish tissues was blocked to significant extents. Consistently, when CsIFIT1 expression in tongue sole was knocked down by siRNA, viral replication in fish tissues was significantly enhanced. Taken together, these results indicate that Cs IFIT1 is likely a key factor of antiviral immunity and is required for optimal defense against viral infection...