文章摘要
苏丹鱼(Leptobarbus hoevenii)甘露糖受体的基因克隆表达和免疫特性研究
Cloning, expression and immune features of Sultan fish ( Leptobarbus hoevenii ) mannose receptor
投稿时间:2019-02-26  修订日期:2019-04-22
DOI:
中文关键词: 苏丹鱼  柱状黄杆菌  甘露糖受体  基因克隆  基因表达
英文关键词: Leptobarbus hoevenii  Flavobacterium columnare  Mannose receptor  Gene cloning  immune responses
基金项目:国家自然科学基金
作者单位E-mail
何昕 广东省水环境与水产品安全工程技术研究中心 1277810700@qq.com 
秦真东 广东省水环境与水产品安全工程技术研究中心  
张凯 广东省水环境与水产品安全工程技术研究中心  
梁日深 广东省水环境与水产品安全工程技术研究中心  
杨森 广东省水环境与水产品安全工程技术研究中心  
伍剑标 佛山市南海区标记鱼苗场  
赵丽娟 广东省水环境与水产品安全工程技术研究中心  
林蠡 广东省水环境与水产品安全工程技术研究中心 linli@zhku.edu.cn 
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中文摘要:
      苏丹鱼(Leptobarbus hoevenii),学名何氏细须鲃,是我国新近从马来西亚引进的名优淡水养殖鱼类。柱状黄杆菌(Flavobacterium columnarium)是苏丹鱼的常见病原,其感染导致其柱形病。研究鱼类感染的免疫特性是开展鱼病高效防控的基础。甘露糖受体(mannose receptor, MR)在鱼类先天性免疫和适应性免疫中都发挥重要作用。为了解苏丹鱼MR(LhMR)的结构特点及其在抗感染免疫反应中的作用,本研究克隆并分析了LhMR基因,采用实时荧光定量PCR(qRT-PCR)技术检测了LhMR 在正常及柱状黄杆菌感染苏丹鱼的组织表达。结果显示,LhMR开放阅读框4296bp,编码1431个氨基酸(aa)。LhMR的氨基酸序列和分子结构与其他物种高度相似:胞外1个富含蓖麻类β型三叶草的结构域(RICIN)、1个纤连蛋白II型结构域(FNII) 、8个串连的C型凝集素样结构域(CLECTs)、1个跨膜区和1个胞内区。通过qRT-PCR检测显示LhMR在脾、体肾、心脏、脑、皮肤、肌肉、鳃 、肝脏、后肠 、前肠 、中肠11种组织中均有表达,其中脾脏表达量最高;经柱状黄杆菌感染苏丹鱼后,脾、肠、肾、鳃LhMR 基因的相对表达量显著升高。通过免疫组织化学(IHC)检测LhMR在脾脏、肾脏、鳃中的MR蛋白表达水平增加。通过苏木精-伊红染色病理组织切片表明在体肾、肠、肝、鳃中都有不同程度的病变。体肾的肾小管有大量的血细胞浸润,上皮细胞发生坏死;肠壁变薄,组织大量弥散坏死;肝组织有多个空泡;鳃小片肿胀、混乱、坏死和脱落。本研究结果表明LhMR在苏丹与感染柱状黄杆菌免疫反应中发挥重要作用,为苏丹鱼柱形病的防控提供新的参考。
英文摘要:
      Sultan fish(Leptobarbus hoevenii)is a freshwater cultured species introduced from Malaysia to China recently. Flavobacterium columnarium is a common pathogen which can infect Sultan fish and causes columnaris. The knowledge about the fish immune responses during the infection is critical for fish disease prevention. There have been reported that mannose receptor (MR) plays a critical role in innate and adaptive immunity of fish. In order to understand the structural characteristics of MR and its role in anti-infective immune responses in Sultan fish, we cloned and sequenced the MR of Sultan fish. Subsequently, real-time polymerase chain reaction (qRT-PCR) assay was used to measure the expression of the MR in the tissues of Sultan fish with or without F. columnare infection. The results showed that the MR of Sultan fish (LhMR) open reading frame was 4296 bp, encoding 1431 amino acids (aa). The amino acid sequence and molecular structure of LhMR are highly similar to the MRs from other animals, i.e., containing an extracellular ricin-like β-type clover domain (RICIN), a fibronectin type II domain (FNII) and eight tandem C Lectin-like domains (CLECTs), a transmembrane region and a short intracellular region. The mRNA of LhMR was widely expressed in 11 tested tissues, including spleen, kidney, heart, brain, skin, muscle, gills, liver, post intestine, fore intestine and mid intestine, with the highest expression level in the spleen. The mRNA expression of LhMR was significantly increased in the spleen, intestine, kidney and gills of Sultan fish infected with F. columnare measured by qRT-PCR. Furthermore, the protein expression of LhMR was also increased in the spleen, kidney and gill of Sultan fish infected with F. columnare revealed by immunochemistry assay. By Hematoxylin eosin staining, there were various pathological changes observed in the trunk kidney, intestine, liver and gills. In the body kidney, there were blood cells infiltrated in the renal tubules with necrosis in epithelial cells. The wall of intestine became thinner with diffused necrosis. Numerous vacuoles were observed in the liver cells. Gill lamella showed swollen, disordered, necrosis and detached. The results revealed that LhMR plays an important role in the immune responses of Sultan fish infected with F. columnare and will pave a new way for development prevention and therapy strategies against columnaris.
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