大黄鱼(Larimichthys crocea)白细胞介素10(IL-10)基因克隆及溶藻弧菌(Vibrio alginolyticus)侵染后表达变化分析

CLONING OF INTERLEUKIN-10(IL-10) GENE FROM LARGE YELLOW CROAKER LARIMICHTHYS CROCEA AND THE EXPRESSION PATTERN ANALYSIS ON VIBRIO ALGINOLYTICUS INFECTION

白细胞介素10(interleukin10,IL-10)是鱼类先天性免疫因子之一,在炎症反应和免疫调节中有着重要作用。本研究获得大黄鱼(Larimichthys crocea)IL-10基因cDNA序列,由899个核苷酸组成,含1个555bp的开放阅读框、编码184个氨基酸,预测编码蛋白分子量为21.24kDa、N端有22个氨基酸组成的信号肽序列。氨基酸序列多重比对表明大黄鱼IL-10具有IL-10家族特征性序列和构成2对二硫键的4个保守性半胱氨酸,与欧洲鲈(Dicentrarchuslabrax)IL-10序列相似性最高(78.5%);系统进化树分析显示大黄鱼IL-10和其他鱼类IL-10形成一个大簇,与欧洲鲈进化关系最近。荧光定量PCR(quantitative real-time PCR, qRT-PCR)检测结果显示健康大黄鱼IL-10基因在鳃和肠中高表达,肝和头肾中低表达;溶藻弧菌(Vibrio alginolyticus)侵染后大黄鱼肠、脑、脾、肝和头肾中IL-10基因表达量均显著上调(P<0.05),其中头肾和肝中上调幅度最大(分别为菌侵染前的10.06倍和8.79倍)。综上,大黄鱼IL-10基因表达与病原菌感染密切相关,为深入研究大黄鱼IL-10的生物学功能及免疫机制提供了基础资料。

In teleost, interleukin 10(IL-10) is an immune factors of the innate immunity system, and it plays an important role in inflammatory responses and immunoregulation. In this study, IL-10 cDNA was cloned and characterized from large yellow croaker Larimichthys crocea. The results showed that L. crocea IL-10 cDNA contained 899 nucleotides with an open reading frame of 555 nucleotides encoding a ploy peptide of 184 amino acids. The molecular mass of L. crocea IL-10 protein was 21.24 kDa, and the 22 amino acid of the N-terminal region formed signal peptide. The multiple sequence alignment of amino acid sequences indicated that L. crocea IL-10 possessed the IL-10 family signature motif, and it had four conserved cysteine residues to form two disulfide bridges. L. crocea IL-10 shared the highest amino acid similarity(78.5%) with IL-10 of European seabass Dicentrarchus labrax. Phylogenetic tree analysis also confirmed that L. crocea IL-10 fell into the fish IL-10 cluster and was most closely related to that of D. labrax. In health L. crocea, quantitative real-time PCR(qRT-PCR) analysis showed that IL-10 mRNA had high expression level in the gill and intestine, and the low expression level was shown in the liver and head kidney. Upon Vibrio alginolyticus infection, L. crocea IL-10 transcripts up-regulated significantly in the intestine, brain, spleen, liver, and head kidney compared to that before the infection(P<0.05). Among these tissues, the head kidney had the most increase amplitude of IL-10 expression(10.06 fold) after V. alginolyticus infection, and followed by the liver(8.79 fold). Therefore, IL-10 gene is closely related to the pathogenic bacteria infection of L. crocea, and these data could be helpful to understand the biological function and immunologic mechanism of L. crocea IL-10 gene.