Abstract:Interleukin 12 (IL-12), a key molecular switch in the immune response, is a pleiotropic cytokine composed of the p35a and p40c subunits. It has been proven that IL-12 can be used as a vaccine adjuvant to enhance the immune response level of fish, and is used in the development of vaccines against pathogen infection. In this study, we cloned the coding regions of the p35a subunit and p40c subunit of IL-12 in Chinese tongue sole (Cynoglossus semilaevis), with a length of 651 bp and 984 bp, respectively. The phylogenetic analysis showed that the p35a and p40c of C. semilaevis were clustered with the corresponding genes in other fishes. The sequence of amino acid homology analysis showed that p35a and p40c of C. semilaevis had the highest similarity with Takifugu rubripes (52.04%) and Gasterosteus aculeatus (48.67%), respectively. The tissue expression analysis showed that p35a was highly expressed in the gill, brain, heart, and ovary, and p40c was highly expressed in the liver, spleen, gill, and heart. We further analyzed the expression patterns of p35a, p40c, and their related genes (ifn-γ and il-10) after infection with Vibrio harveyi. The expression of p35a increased significantly at 48 h (P<0.05), and then gradually decreased in the spleen, and its expression increased significantly at 72 h in the liver and intestine. The expression of p40c increased significantly at 6 h in spleen and liver, and at 48 h in kidney. Its expression also began to increase at 6 h and peaked at 48 h in the intestine. The expression of ifn-γ increased significantly before the upregulated expression of p35a in the liver and intestine, and later than p35a in the spleen. The expression of il-10 was opposite to that of p40c in the liver, spleen, kidney, and intestine. The expression of the immune related cytokines (tnf-α, tnf-β, ifn-γ, and il-10) in the lymphocytes of C. semilaevis was detected after the overexpression of the p35a and p40c genes. The results showed that p35a and p40c could significantly increase the expression of tnf-α under lipopolysaccharide stimulation. The results indicate that the p35a and p40c subunits of IL-12 respond to stimulation by V. harveyi in C. semilaevis, and then participate in the immune response by inducing the expression of the tnf-α gene, which provides a theoretical basis for the development of IL-12 as an adjuvant for the C. semilaevis vaccine against V. harveyi.
