Molecular identification and expression analysis of the natural killer cell enhancing factor (NKEF) gene from turbot ( Scophthalmus maximus)

Abstract

Natural killer cell enhancing factor (NKEF) belongs to the newly defined peroxiredoxin (Prx) family. It was originally isolated from human red blood cells. It has the ability to enhance the cytotoxic activity of natural killer cells, and appears most effective in the protection of DNA and protein against oxidative damage, chemotherapy agents, and inflammation-induced monocyte adhesion. However, few reports are available about the structure and function of NKEF in lower vertebrates such as teleosts. We have recently isolated a cDNA encoding NKEF from turbot ( Scophthalmus maximus). The cDNAs of NKEF were cloned from turbot spleen cDNA library. The 904 bp full-length cDNA of the NKEF was obtained by RACE and consisted of 122 bp 5′ terminal UTR, 591 bp encoding region and 191 bp 3′ terminal UTR. The 591 bp open reading frame was found to code for a protein with 197 amino acid residues. The deduced amino acid sequence of turbot had 85.3%, 81.7%, 81.2%, 80.7%, 80.7%, 79.2%, 78.2%, 77.7%, 77.7%, 77.2% and 58.2% identity with that of zebrafish, channel catfish, flounder, Tetraodon nigroviridis, rainbow trout and mouse Prx2-1, human NKEF-A, carp and rat TSA, human NKEF-B and yeast TSA, respectively. RT-PCR indicated that NKEF transcripts were abundant in the head kidney, spleen and liver, less abundant in the kidney and intestine, and least in gill, muscle and heart. The level of NKEF mRNA in embryos gradually increases during embryogenesis from two cell stage to fry stage. Challenge of turbot with pathogenic bacteria Vibrio anguillarum, significantly elevated NKEF mRNA levels in the liver and spleen in a time-dependent fashion. The NKEF transcripts were detected in turbot embryonic cell line (TEC). Challenge of the TEC cell cultures with pathogenic bacteria, V. anguillarum, significantly elevated NKEF mRNA levels in TEC cell cultures 48 h after infection.