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Molecular cloning of Japanese eel Anguilla japonica TNF-α and characterization of its expression in response to LPS, poly I:C and Aeromonas hydrophila infection

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Chinese Journal of Oceanology and Limnology 2014年第5期32卷 1046-1059页

作者：冯建军关瑞章郭松林林鹏 frank zadlock College of Fisheries Jimei University Engineering Research Center of Eel Modern Industry Technology Ministry of Education Key Laboratory of Healthy Mariculture for the East China Sea Ministry of Agriculture Department of Biology Seton Hall University

As a potent pleiotropic cytokine, tumor necrosis factor-alpha （TNF-ct） plays an important role in innate immune responses. The cDNA sequence and genomic structure of the TNF-α gene （AjTNF-c0 in the Japanese eel （Anguilla japonica） were identified and characterized. The full-length AjTNF-α cDNA was 1 546 bp, including a 5＇-untranslated region （UTR） of 13 bp, a 3＇-UTR of 879 bp and an open reading frame of 654 bp encoding a protein of 218 amino acids. The full-length genomic sequence of AjTNF-ct was 2 392 bp and included four exons and three introns. The putative AjTNF-α protein contained TNF family signature motifs, including a protease cleavage site, a transmembrane domain and two conserved cysteine residues. Quantitative real-time reverse transcription PCR analysis revealed AjTNF-α expression in a wide range of tissues, with predominant expression in blood and liver. Lower levels of expression were seen in spleen, gills, kidney, intestine, heart, and skin, with very low levels in muscle. The modulation of AjTNF-ct expression after injection of eels with lipopolysaccharide （LPS）, the viral mimic, poly I：C, or Aeromonas hydrophila was assessed in blood, liver, and kidney. In blood, TNF-α mRNA levels increased rapidly and then rapidly decreased after stimulation with LPS, poly I：C or A. hydrophila. However, the response to LPS and A. hydrophila peaked at 6 h while for poly I：C the peak was at 12 h. In liver, after injection with A. hydrophila, an up- and down-regulation of AjTNF-ct expression occurred twice, peaking at 6 h and 24 h, respectively. No remarkable increase of AjTNF-α expression appeared in liver until 72 h after LPS or poly I：C treatment. In kidney, ApiNF-α expression increased significantly only at 72 h post-stimulation with LPS orA. hydrophila. Our results suggest that AjTNF-α plays an important role in fish in the defense against viral and bacterial infection.

关键词： tumor necrosis factor-alpha Anguilla Japonica cDNA mRNA expression immune response

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