半滑舌鳎白细胞介素12的p35a和p40c亚基在哈维氏弧菌感染下的表达和调控分析

Expression and Regulation Analysis of the p35a and p40c Subunits of Interleukin 12 in Cynoglossus semilaevis Infected by Vibrio harveyi

关键词:  半滑舌鳎  免疫应答  白细胞介素12  哈维氏弧菌

Key words:  Cynoglossus semilaevis  Immune response  Interleukin 12  Vibrio harveyi

摘要:

本研究以海水养殖鱼类半滑舌鳎(Cynoglossus semilaevis)为研究对象，克隆了白细胞介素12 (IL-12)的p35a亚基和p40c亚基的基因编码区序列，编码区长度分别为651 bp和984 bp。系统进化树分析显示，半滑舌鳎的p35a和p40c分别与其他鱼类对应的基因聚为一支。氨基酸同源性分析显示，p35a与红鳍东方鲀(Takifugu rubripes)相似性最高，p40c与三棘刺鱼(Gasterosteus aculeatus)相似性最高，分别为52.04%和48.67%。组织表达分析显示，p35a在鳃、脑、心和卵巢中表达量较高，p40c在肝、脾、鳃和心中表达量较高。通过哈维氏弧菌(Vibrio harveyi)感染实验，分析了半滑舌鳎p35a、p40c及其相关基因(ifn-γ和il-10)在哈维氏弧菌感染过程中的表达模式，p35a在脾中，48 h表达显著上升(P<0.05)，之后表达逐渐下降；在肝和肠中，72 h表达显著上升。p40c在脾、肝中，6 h表达显著上升；在肾中，48 h产生上调；在肠中，6 h开始上调，并在48 h上升至最高点。ifn-γ在肝和肠中，均在p35a表达上调之前显著升高，在脾中晚于p35a上调表达；il-10在肝、脾、肾、肠中表达趋势中均与p40c的表达趋势相反。将半滑舌鳎淋巴细胞过表达p35a和p40c基因后，检测了受不同浓度脂多糖(LPS)刺激后干扰素(ifn-γ)、肿瘤坏死因子(tnf-α和tnf-β)等免疫相关细胞因子的表达变化。结果显示，p35a、p40c能够显著提高tnf-α在LPS刺激下的表达水平。上述结果表明，IL-12的p35a亚基和p40c亚基能够响应哈维氏弧菌对半滑舌鳎的刺激，过表达p35a、p40c能够通过诱导tnf-α基因的表达来参与机体的免疫应答。研究结果可为IL-12作为半滑舌鳎抗哈维氏弧菌疫苗佐剂的开发提供理论基础。

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.