大黄鱼(Larimichthys crocea)补体调节因子CFH和CFHR2基因的分子特征及表达分析

CHARACTERIZATION AND EXPRESSION ANALYSIS OF COMPLEMENT REGULATOR FACTOR CFH AND CFHR2 IN LARGE YELLOW CROAKER LARIMICHTHYS CROCEA

补体是鱼类免疫系统重要的组分,具有识别和消除外来病原体,激活免疫细胞,调控获得性免疫等功能。在免疫组织中,补体调节因子大约占据了补体成分的二分之一,当受到外界病原刺激时会迅速活化补体成分,并且进一步聚合形成酶复合物发挥一系列免疫效应。CFH和CFHR2是补体替代途径重要的调节因子,对于补体系统正常运转必不可少。为此,本文测定了大黄鱼补体调节因子CFH和CFHR2基因的c DNA全序列,并对基因组织特异性表达和溶藻弧菌刺激后基因m RNA表达量的变化等方面进行了研究。CFH和CFHR2序列全长分别为1332 bp和1170 bp,分别编码443和389个氨基酸,N端信号肽序列分别为24和32个氨基酸。推导的氨基酸序列结构分析表明大黄鱼CFH和CFHR2基因具有RCA蛋白家族的典型特征,即含有多个保守的CCP结构(补体控制蛋白)。实时荧光定量PCR结果显示,CFH和CFHR2在健康大黄鱼的肝、脾、肾、肠、脑、胃、心和肌肉这8种组织中都有表达,其中肝脏的表达量显著高于其它几种组织。溶藻弧菌(Vibrio alginolyticus)侵染了健康的大黄鱼之后,CFH和CFHR2的m RNA表达量均明显上调,并且随着感染时间的变化呈现不同的上升趋势。结果表明补体调节因子m RNA表达量的变化与溶藻弧菌的侵染密切相关,表明了CFH和CFHR2可能在大黄鱼自身免疫机制中发挥重要的作用。

The complement system is an important component in fish innate immunity which can recognize, facilitate the elimination of pathogens, promote the function of phagocyte, activate immune cell as well as modulate the adaptive immune response. In the immune tissues, about half of the complement components exist as regulating factors, which play key role in complement activation. CFH and CFHR2 are important regulator factors in alternative pathways of complement system and maintain component system function well. Although the complement system of Larimichthys crocea has been extensively studied, the expression and evolution of CFH and CFHR2 remain unknown. In this study, we reported the primary sequence of Larimichthys crocea complement regulator factor H and HR2. CFH open reading frame（ORF） is 1332 bp and encodes for 443 amino acids with a putative signal peptide of 24 amino acid residues. CFHR2 open reading frame（ORF） is 1170 bp and encodes for 339 aa with a putative signal peptide of 32 amino acid residues. Deduced amino acid sequence of the structure analysis indicated that the L. crocea CFH and CFHR2 have typical characteristics of RCA protein family, possessing several conservative CCP structures（complement control protein）. Real-time PCR analysis showed that the Liver displayed the most abundant expression level of three genes among the tested tissues of the healthy individuals. We found that Vibrio alginolyticus exposure up-regulated the expressions significantly in the early induction. As the change of infection time, the expression present different rising trend. The result showed that CFH and CFHR2 gene are very sensitive to pathogenic substances, which indicated that complement regulator factors may play an significant role in immunologic mechanisms of croaker.