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

【摘要】 As a potent pleiotropic cytokine, tumor necrosis factor-alpha(TNF-α) plays an important role in innate immune responses. The cDNA sequence and genomic structure of the TNF-α gene( Aj TNF-α) in the Japanese eel( Anguilla japonica) were identified and characterized. The full-length Aj TNF-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 Aj TNF-α was 2 392 bp and included four exons and three introns. The putative Aj TNF-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 Aj TNF-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 Aj TNF-α 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 Aj TNF-α expression occurred twice, peaking at 6 h and 24 h, respectively. No remarkable increase of Aj TNF-α expression appeared in liver until 72 h after LPS or poly I:C treatment. In kidney, Aj TNF-α expression increased significantly only at 72 h post-stimulation with LPS or A. hydrophila. Our results suggest that Aj TNF-α plays an important role in fish in the defense against viral and bacterial infection.更多还原

【Abstract】 As a potent pleiotropic cytokine, tumor necrosis factor-alpha(TNF-α) plays an important role in innate immune responses. The cDNA sequence and genomic structure of the TNF-α gene( Aj TNF-α) in the Japanese eel( Anguilla japonica) were identified and characterized. The full-length Aj TNF-α cDNA was 1546 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 Aj TNF-α was 2 392 bp and included four exons and three introns. The putative Aj TNF-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 Aj TNF-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 Aj TNF-α 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 Aj TNF-α expression occurred twice, peaking at 6 h and 24 h, respectively. No remarkable increase of Aj TNF-α expression appeared in liver until 72 h after LPS or poly I:C treatment. In kidney, Aj TNF-α expression increased significantly only at 72 h post-stimulation with LPS or A. hydrophila. Our results suggest that Aj TNF-α plays an important role in fish in the defense against viral and bacterial infection.更多还原