鱼类病原性弧菌血清学诊断芯片技术构建  

Construction of Serological Diagnostic Chip for Pathogenic Vibrio in Fish

作　　者：绳秀珍[1] 李文涛[1] 王欣欣[1] 唐小千[1] 邢婧[1] 战文斌[1,2] Sheng Xiuzhen;Li Wentao;Wang Xinxin;Tang Xiaoqian;Xing Jing;Zhan Wenbin(Laboratory of Pathology and Immunology of Aquatic Animals,Ocean University of China,Qingdao,Shandong 266003,China;Function Laboratory for Marine Fisheries Science and Food Production Processes,Qingdao National Laboratory for Marine Science and Technology,Qingdao,Shandong 266071,China)

机构地区：[1]中国海洋大学水产动物病害与免疫学实验室,山东青岛266003 [2]青岛海洋科学与技术国家实验室海洋渔业科学与食物产出过程功能实验室,山东青岛266071

出　　处：《中国动物检疫》2019年第12期88-97,共10页China Animal Health Inspection

基　　金：国家重点研发计划“蓝色粮仓科技创新”重点专项（2019YFD0900101）;国家自然科学基金项目（31872599,31730101,31672685）;山东省泰山学者特聘专家计划项目

摘　　要：针对鱼类致病性肠道弧菌(Vibrio ichthyoenteri)、鳗弧菌(Vibrio anguillarum)、副溶血弧菌(Vibrio parahaemolyticus)、溶藻弧菌(Vibrio alginolyticus)、河流弧菌(Vibrio fluvialis)和哈维氏弧菌(Vibrio harveyi),制备了牙鲆(Paralichthys olivaceus)抗血清,提取了各弧菌的外膜蛋白、胞外产物、鞭毛蛋白和全菌蛋白,分析了牙鲆抗血清与各抗原蛋白的免疫反应,发现各弧菌抗血清与各自的抗原反应最强,与其他弧菌抗原有不同程度的交叉反应。将6种弧菌的抗原组分固定于芯片载体形成微阵列,构建了其血清学诊断抗原芯片,使用辣根过氧化物酶标记的抗牙鲆IgM单抗2D8作为检测抗体,确定了检测结果判读方法;将抗原芯片应用于牙鲆和大菱鲆(Scophthalmus maximus)的弧菌病诊断,并利用ELISA技术,验证了该抗原芯片用于弧菌病诊断的准确性。本研究为鱼类弧菌病的血清学诊断提供了有力工具。The antiserum samples of Paralichthys olivaceus were prepared respectively against 6 kinds of pathogenic vibrios,including Vibrio ichthyoenteri,V.anguillarum,V.parahaemolyticus,V.alginolyticus,V.fluvialis and V.harveyi.The outer membrane protein,flagellum protein,extracellular products and whole-cell bacterial protein of the strains were extracted and purified,then the immune reactions between the antigen components and the antiserum samples were analyzed,it was found that,for all Vibrios,the immune reaction between its antigen and its own antiserum was the strongest,besides,there were also cross-reactions in different extent between the antiserum with the antigen of other Vibrios.Further,the antigen components of the 6 strains were fixed on the chip carrier to form an antigen microarray so as to construct the serological diagnostic antigen chip,the method for interpreting detection results was determined by using monoclonal antibody 2D8 of IgM marked with horseradish peroxidase as the detection antibody;the antigen chip was applied to diagnose vibriosis in Paralichthys olivaceus and Scophthalmus maximus,and its accuracy was verified by ELISA.As a conclusion,a useful tool was provided for serological diagnosis of fish vibriosis.

关 键 词：弧菌 抗原芯片 血清学诊断 交叉反应 ELISA 牙鲆 

分 类 号：S852.53[农业科学—基础兽医学]