Molecular cloning and characterization of IKKε gene from black carp Mylopharyngodon piceus

Abstract

IKKε is an IκB kinase functioning in NF-κB signal pathway in the innate immune system of higher vertebrates. To exploit the function of IKKε of black carp (bcIKKε) in its antiviral innate immunity, the IKKε gene has been cloned from the RNA isolated from the spleen of black carp. The full-length cDNA of bcIKKε is 2537 bp, which encodes the peptide of 723 amino acids. bcIKKε contains a S-Tkc domain, a PKc domain and a UBL-TBK1-like domain and bcIKKε shares the highest amino acid sequence similarity with that of grass carp. bcIKKε was constitutively transcribed in the selected tissues of black carp including gill, kidney, heart, intestine, liver, muscle, skin and spleen; and the mRNA level of bcIKKε in these tissues varied right after SVCV or GCRV infection. bcIKKε had been well expressed in HEK293T cells and western blot assay determined that this fish kinase was around 80 KDa. The immunofluorescence assay of both NH3T3 cells and EPC cells demonstrated that bcIKKε was located in the cytosolic part of the cell. Report assay result showed that overexpression of bcIKKε in EPC cells activated the expression of both zebrafish IFN and EPC IFN. All our data suggest that bcIKKε is a novel fish kinase functioning in the innate antiviral immune response of black carp.

Keywords: Black carp; IKKε; Innate immunity.