Molecular cloning, expression analysis and immune-related functional identification of tumor necrosis factor alpha...

Tumor necrosis factor α (TNFα), a cytokine mainly secreted by active macrophages and monocytes, causes hemorrhagic necrosis of tumor tissues, kills tumor cells, regulates inflammatory responses, and plays a crucial role in innate immunity. In this study, TNFα of Sepiella japonica (named as SjTNFα) was acquired, whose full-length cDNA was 1206 bp (GenBank accession no. ON357428), containing a 5' UTR of 185 bp, a 3' UTR of 137 bp and an open reading frame (ORF) of 1002bp to encode a putative peptide of 333 amino acids for constructing the transmembrane domain and the cytoplasmic TNF domain. Its predicted pI was 8.69 and the theoretical molecular weight was 44.72 KDa. Multiple sequence alignment and phylogenetic analysis showed that SjTNFα had the highest homology to Octopus sinensis, they fell into a unified branch and further clustered with other animals. Real-time PCR indicated that SjTNFα was widely expressed in all subject tissues, including spleen, pancreas, gill, heart, brain, optic lobe, liver and intestine, and exhibited the highest in the liver and the lowest in the brain. The relative expression of SjTNFα varied at the developmental period of juvenile stage, pre-spawning and oviposition in the squid, with the highest in the liver at the juvenile stage and oviposition, and in the optic lobe of pre-spawning. After being infected with Vibrio parahaemolyticus and Aeromonas hydrophila, the expression of SjTNFα in liver and gill were both upregulated with time, and the highest expression appeared at 24 h and 8 h in liver for different infection, and at 4 h in gill consistently. Cell localization showed that SjTNFα distributed on membrane of HEK293 cells because it was a type II soluble transmembrane protein. When HEK293 cells were stimulated with LPS of different concentrations, the NF-κB pathway was activated in the nucleus and the corresponding mRNA was transferred through the intracellular signal transduction pathway, resulting in the synthesis and release of TNFα, which made the expression of SjTNFα was up-regulated obviously. These findings showed that SjTNFα might play an essential role in the defense of S. japonica against bacteria challenge, which contributed to the understanding of the intrinsic immune signaling pathway of Cephalopoda and the further study of host-pathogen interactions.