黄颡鱼甘露糖受体基因的克隆和功能分析  

Cloning and functional analysis of mannose receptor from Chinese yellow catfish(Pelteobagrus fulvidraco)

作　　者：刘小玲[1,2] 王虹[1,2] 樊启学[1,2] 兰江风[1,2,3] 林蠡[1,2,3,4] LIU Xiaoling WANG Hong FAN Qixue LAN Jiangfeng LIN Li(Department of Aquatic Animal Medicine, College of Fisheries, Huazhong Agricultural University, Wuhan 430070, China College of Fisheries, Huazhong Agricultural University, Freshwater Aquaculture Collaborative Innovation Center of Hubei Province, Wuhan 430070, China College of Fisheries, Huazhong Agricultural University, Hubei Provincial Engineering Laboratory for Pond Aquaculture, Wuhan 430070, China Key Lab of Freshwater Animal Breeding, College of Fisheries, Huazhong Agricultural University, Ministry of Agriculture, Wuhan 430070, China)

机构地区：[1]华中农业大学水产学院,水生动物医学系,湖北武汉430070 [2]华中农业大学水产学院,淡水水产健康养殖湖北省协同创新中心,湖北武汉430070 [3]华中农业大学水产学院,池塘健康养殖湖北省工程实验室,湖北武汉430070 [4]华中农业大学水产学院,农业部淡水生物繁育重点实验室,湖北武汉430070

出　　处：《水产学报》2017年第7期1036-1043,共8页Journal of Fisheries of China

基　　金：国家自然科学基金(31372563,31572657,31502199);中央高校基本科研业务费专项(2014PY035,2662015QC019);湖北省科技支撑计划(2015BBA228,YSF2015000255);农业部渔用药物创制重点实验室开放课题(201401);广东省海洋与渔业局基金(A201512C003,2015-115);武汉市科技局基金(2016020101010089)~~

摘　　要：甘露糖受体(MR)隶属凝集素超家族,主要表达于巨噬细胞和未成熟的树突状细胞表面。MR不仅在先天免疫防御中发挥重要作用,还通过参与抗原呈递,激活T淋巴细胞,启动获得性免疫应答过程。本研究采用同源克隆技术获得黄颡鱼甘露糖受体(pf MR)基因,采用荧光定量PCR技术检测MR在正常黄颡鱼体内的分布情况,采用鲖爱德华菌感染黄颡鱼头肾巨噬细胞和甘露聚糖封闭MR方法研究黄颡鱼MR在抗细菌感染中的作用。结果显示,pf MR同团头鲂、草鱼、斑马鱼和尼罗罗非鱼的MR聚为一支。pf MR在所检测的12个组织中均有分布,其在肾脏、脾脏和肌肉组织中表达量较高,在血液中表达量较少。鲖爱德华菌感染黄颡鱼头肾巨噬细胞后,pf MR、IL-1β和TNF-a均被细菌诱导表达,超氧阴离子和一氧化氮的含量也上升,超氧阴离子在感染30 min后即显著上升,一氧化氮在感染12 h后才显著上升。甘露聚糖竞争结合MR,显著抑制巨噬细胞内化GFP标签的鲖爱德华菌,加入EDTA减少内化的荧光强度,加入Ca^(2+)使内化的荧光强度回升。研究表明,黄颡鱼头肾巨噬细胞MR参与鲖爱德华菌的识别和内吞过程,而且依赖Ca^(2+)。Mannose receptor（MR） is a member of C-lectin family. It is mainly expressed on the surface of macrophage and immature dendritic cells. It has been shown that MR not only plays a significant role in innate immune responses, but also initiates acquired immune responses through processing and presenting antigens to activate T cells. In this study, we cloned and characterized pf MR of Chinese yellow catfish（Pelteobagrus fulvidraco）. The expression profiles of pf MR mRNA in different tissues of P. fulvidraco were assessed by real-time quantitative reverse transcription PCR（qRT-PCR）. To investigate the role of pf MR participating in the antibacterial infection, yellow catfish head kidney macrophages were infected with Edwardsiella ictaluri and competition binding test of mannan to MR was carried out. The phylogenetic tree analysis showed that MR of P. fulvidraco,Megalobrama amblycephala, Ctenopharyngodon idella, Danio rerio, and Oreochromis niloticus formed a clade.The mRNA of pf MR was expressed in all detected tissues with higher level in the kidney, spleen and muscle, and lowest expression in the blood. The expressions of pf MR, IL-1β and TNF-a mRNAs were all increased in cultured primary head-kidney macrophages after E. ictaluri infection. The infection of E. ictaluri also resulted in the overexpression of large number of superoxide anion（O_2^-·） and nitric oxide（NO） in the macrophages. The infection of E. ictaluri could significantly activate the production ofO_2^-· at 30 min post treatment, while the amount of NO in the macrophage was significantly elevated at 12 h post treatment. Mannan significantly inhibited the engulfed GFP-E. ictaluri by macrophages through competition binding to MR. The engulfed GFP-E. ictaluri was significantly reduced by addition of EDTA, and restored by addition of Ca^（2＋）. The present study indicated that MR in head kidney macrophages of yellow catfish mediated phagocytosis of E. ictaluri and it was Ca^（2＋）-dependent.

关 键 词：黄颡鱼 甘露糖受体 鲖爱德华菌 超氧阴离子 一氧化氮 

分 类 号：Q785[生物学—分子生物学] S965[农业科学—水产养殖]