First molluscan TNF-alpha homologue of the TNF superfamily in disk abalone: molecular characterization and expression analysis.

Tumor necrosis factor alpha (TNF-alpha) is considered as a multifunctional immune modulator that plays an important role in the innate and adaptive immune systems in vertebrates. Here, we described the characterization and expression analysis of the first TNF-alpha homologue in mollusk abalone, named as AbTNF-alpha. It has 930-bp full length with a 717-bp open reading frame (ORF), encoding 239 amino acids. The AbTNF-alpha amino acid sequence shows the characteristic TNF family signature, N-terminal transmembrane domain consisting of a hydrophobic amino acid cluster and cell attachment sequence at (155)RGD(157). Phylogenic analysis results showed that AbTNF-alpha is more related to the invertebrate Ciona savignyi TNF superfamily ligand member (CsTL). Quantitative real-time PCR expression results showed that AbTNF-alpha was constitutively expressed in both immune and non-immune tissues in a tissue specific manner. The highest constitutive expression was in the gill tissue with a 1.5-fold compared to hemocytes expression. The AbTNF-alpha mRNA expression in gill tissue was monitored in vivo stimulated by a mixture of pathogenic bacteria (Vibrio alginolyticus, Vibrio parahemolyticus, and Lysteria monocytogenes), viral haemorrhagic septicaemia virus (VHSV) and lipopolysaccharide (LPS). The AbTNF-alpha expression was significantly (p<0.05) induced by bacteria, VHSV and LPS compared to the control animals. Moreover, the highest level expressions of each induction were at 24 h (5.2-fold), 48 h (2.8-fold), and 48 h (3.3-fold) by bacteria mixture, VHSV and LPS, respectively. These results indicate that AbTNF-alpha could respond to pathogenic infection or stimulation and may play an important role in the abalone immune system.