Functional Characterization of TNFα in the Starry Flounder (Platichthys stellatus) and Its Potential as an Immunostimulant

Abstract

Tumor necrosis factor alpha (TNF alpha) is a central pro-inflammatory cytokine that mediates host immune responses during infection. In this study, we identified and characterized the TNF alpha gene in the starry flounder (Platichthys stellatus) through transcriptomic analysis. The deduced protein contained a conserved TNF domain and transmembrane region, and phylogenetic analysis confirmed its homology with other teleost TNF alpha proteins. Tissue-specific expression profiling revealed high baseline expression in immune-related peripheral organs and a distinct temporal modulation in response to Streptococcus parauberis infection. Recombinant TNF alpha (rTNF alpha), produced using a cell-free expression system, significantly enhanced phagocytic activity in peripheral and kidney-derived leukocytes in a dose-dependent manner. Peak activity was observed at 150-200 mu g/mL, while a decline at higher concentrations suggested a threshold for immune stimulation. Importantly, hemolysis assays confirmed the safety of rTNF alpha even at the highest tested concentrations. These results demonstrate the immunomodulatory potential of TNF alpha as a molecular adjuvant in aquaculture vaccines and underscore its potential utility in immune-enhancing strategies for sustainable aquaculture.