[Molecular cloning and expression patterns of two tumor necrosis factor al pha genes in Crucian carp (Carassius...

Tumor necrosis factor (TNF) is a potent inflammatory cytokine produced during inflammation. In this study, two Crucian carp TNF-alpha genes, TNFalpha-1 and TNFalpha-2 were cloned and sequenced. The TNFalpha-1 was 720 bp and-consisted of a 699 bp opening reading frame encoding 232 amino acids and TNFalpha-2 was 793 bp and contained an open reading frame of 687 bp which encoded 228 amino acids. The genomic structure of both genes consists of4 exons and 3 introns similar to other known TNF-alpha genes. The amino acid sequence ofcrucian carp TNFalpha-1 and TNFalpha-2 shared the highest identity with common carp, goldfish and the lowest with flounder and human. The phylogenetic analysis grouped crucian carp TNFalpha-1 and TNFalpha-2 with other cypriniformes, which are more closely related to the goldfish and common carp TNFEa-1 and TNFalpha-2. Real time PCR analysis showed a con- stitutive expression of crucian carp TNFalpha-1 and TNFalpha-2 in all seven tissues examined. The TNFalpha-1 mRNA was ex- pressed significantly higher in the liver and kidney than those of TNFalpha-2 while TNFalpha-2 was expressed significantly higher in the muscle than that of TNFalpha-1. Aeromonas hydrophila BSK-10 strain upregulated the expression level of TNFalpha-1 and TNFalpha-2 in all tissues tested. At 6 h, the expression level of TNFalpha-1 increased significantly higher in muscle, skin and liver, while the expression level of TNFalpha-2 increased significantly higher in muscle and gill. TNFalpha-1 expressed much stronger than TNFalpha-2. At 12 h the expression level started declining and was more reduced at 24 h. These results imply that both TNFalpha-1 and TNFalpha-2 mRNA are distributed differently in tissues and are implicated in the immune response to bacterial infection.