Molecular cloning and characterization of two types of IκBα orthologues in orange-spotted grouper, Epinephelus coioides

Inhibitors of kappa B (IκBs) are the members of primary regulators of NF-κB, which can inhibit NF-κB activity by blocking the NF-κB in an inactive state in the cytoplasm. In this study, two types of IκBα orthologues (EcIκBαA and EcIκBαB) from orange-spotted grouper, Epinephelus coioides, were cloned and characterized. EcIκBαA and EcIκBαB encoded putative proteins containing 308 and 318 amino acids, which shared 59% and 53% identity to IκBαA and IκBαB of Danio rerio, respectively. Amino acid sequence alignment showed that both EcIκBαA and EcIκBαB contained a conserved degradation motif DSGLDS in the N-terminal region and a PEST sequence in the C-terminal region. In addition, EcIκBαA and EcIκBαB contained 5 and 6 ankyrin repeats, respectively. The genomic DNA of EcIκBαA and EcIκBαB consisted of 6 exons and 5 introns. Both of their transcripts were widely distributed in different tissues, and the expression levels were different in response to various stimuli, including lipopolysaccharide (LPS), Vibrio alginolyticus and Singapore grouper iridovirus (SGIV). Dual-luciferase reporter assay suggested that both EcIκBαA and EcIκBαB were able to inhibit Ecc-Rel and Ecp65 induced NF-κB promoter activity in grouper spleen (GS) cells. Subcellular localization analysis showed that EcIκBαB was present predominantly in the cytoplasm, while EcIκBαA was distributed throughout both the nucleus and the cytoplasm. Furthermore, overexpression of EcIκBαA and EcIκBαB in GS cells inhibited the viral gene transcriptions of MCP, ORF019 and ORF162 of SGIV. Taken together, our findings suggested that both EcIκBαA and EcIκBαB were involved in grouper innate immunity against virus.