Immune responses, subcellular localization, and antiviral activity of interferon-induced protein 35 (IFP35) in rock bream (Oplegnathus fasciatus).

Interferon-induced protein 35 kDa (IFP35) has been demonstrated to play important roles in antiviral defense, inflammatory response and cancer progression. However, its precise function in teleost fish remains to be elucidated. Herein, we functionally characterized the rock bream (Oplegnathus fasciatus) IFP35 (OfIFP35) to understand its expression pattern, subcellular localization, antiviral activity, and regulation of downstream genes. OfIFP35 consists of an 1107 bp open reading frame encoding 368 amino acids, including two N-myc-interactor (Nmi)/IFP35 domains (NIDs). The predicted molecular weight of OfIFP35 was 42 kDa, with a theoretical isoelectric point (pI) of 5.10. Evolutionary conservation of IFP35 was analyzed using multiple, pairwise alignments and phylogenetic tree analysis. OfIFP35 in rock bream was found to be highest expressed in the gills. Immune challenges with iridovirus, polyinosinic:polycytidylic acid, lipopolysaccharide, and live bacteria (Streptococcus iniae and Edwardsiella tarda) significantly upregulated its mRNA expression in gill and liver tissues of the rock bream. GFP-tagged OfIFP35 was localized in the cytoplasm of FHM cells, and its overexpression significantly suppressed VHSV transcription in vitro. Moreover, the analysis of downstream gene expression revealed that OfIFP35 could activate the type I interferon pathway. Collectively, these findings indicate that OfIFP35 is important for the immune system of rock bream as it promotes defense responses during viral infections.

Transcriptional modifications and the cytoprotective, DNA protective, and wound healing effects of peroxiredoxin-1 from Sebastes schlegelii.

Peroxiredoxins are a group of thiol-specific antioxidant proteins that take six isoforms in vertebrates and allow the innate immune system to sense and detoxify reactive oxygen species. In this study, we identified and characterized the perxiredoxin-1 (SsPrdx1) cDNA sequence from the rockfish, Sebastes schlegelii. In silico analysis revealed that SsPrdx1 contained a 594 bp long open reading frame (ORF) encoding a protein of 198 amino acids, with a predicted molecular weight and theoretical isoelectric point of 21.97 kDa and 6.30, respectively. The SsPrdx1 gene comprised six exons linked by five introns, while peroxiredoxin signature motifs were found in the highly conserved third, fourth, and fifth exons. Phylogenetic analysis and sequence alignment suggested that SsPrdx1 is evolutionarily conserved and that its most closely related counterpart is Salarias fasciatus. Recombinant SsPrdx1 (rSsPrdx1) displayed supercoiled DNA protection and insulin disulfide reduction activities in a concentration-dependent manner, while cells transiently transfected with pcDNA3.1 (+)/SsPrdx1 exhibited significant cytoprotective effects under oxidative stress and wound healing activity. SsPrdx1 transcripts were constitutively expressed under normal physiological conditions, with the highest expression observed in the blood. Moreover, SsPrdx1 expression increased in the blood, spleen, and liver following immune provocation by LPS, poly I:C, and Streptococcus iniae injection. Thus, this study provides insights into the role of SsPrdx1 in rockfish immune protection.

Molecular expression analysis and characterization of rockfish (Sebastes schlegelii) B cell activating factor.

B cell activating factor (BAFF) is recognized as a member of the TNF superfamily proteins that mediate the immune responses. In this study, BAFF from rockfish (Sebastes schlegelii) (SsBAFF) was characterized based on its functional aspects. The open reading frame of SsBAFF is 804 bp in length and encodes a 267 long amino acid residue protein with predicted molecular weight of 29.48 kDa. The deduced protein sequence comprises with transmembrane domain, furin cleavage site and TNF domain carrying Flap binding site that unique to TNF family. Recombinant SsBAFF (rSsBAFF) significantly enhanced rockfish lymphocytes proliferation and viability in a concentration dependent-manner according to the results from water soluble tetrazolium salt (WST-1) assay and flow cytometric assay. rSsBAFF also modulated the expression of genes involved in anti-inflammatory (IL-10 and NFκB-2) and anti-apoptotic (Bcl-2 and Bax) signal pathways. SsBAFF mRNA expression was detected ubiquitously in all analyzed rockfish tissues, with the highest levels in the spleen and head kidney. Further, the expression of SsBAFF in spleen were significantly induced following LPS, poly (I:C) and Streptococcus iniae challenges. These findings strongly suggest that SsBAFF might play an important role in rockfish immune system through regulating the inflammatory response and proliferation of immune cells.