鲤疱疹病毒Ⅱ型ORF72单克隆抗体的制备及应用  

Preparation and Application of Monoclonal Antibodies against ORF72 of Cyprinid Herpesvirus 2

作　　者：徐超楠 孙雨雨 王佳 郭宝琴 魏畅 李强 XU Chaonan;SUN Yuyu;WANG Jia;GUO Baoqin;WEI Chang;LI Qiang(Key Laboratory of Mariculture&Stock Enhancement in North China′s Sea,Ministry of Agriculture and Rural Affairs,Dalian Ocean University,Dalian 116023,China;Department of Ocean Technology,College of Marine and Biology Engineering,Yancheng Institute of Technology,Yancheng 224051,China)

机构地区：[1]大连海洋大学水产与生命学院,辽宁大连116023 [2]盐城工学院海洋与生物工程学院,江苏盐城224051

出　　处：《水产科学》2024年第4期598-605,共8页Fisheries Science

基　　金：国家自然科学基金资助项目(32102849);江苏省重点研发计划(BE2021326)。

摘　　要：鲤疱疹病毒Ⅱ型是引起鲫造血器官坏死病的重要病原。为建立基于抗原水平的免疫检测方法,通过原核表达方法制备鲤疱疹病毒Ⅱ型核衣壳蛋白ORF72,将纯化的ORF72蛋白免疫BALB/c小鼠,应用杂交瘤技术筛选获得单克隆抗体1B3、4B1、4C2、5F4和5H9。免疫印迹分析显示,上述5株单抗均可特异性识别重组蛋白ORF72;免疫荧光分析显示,仅单抗4C2可与感染鲤疱疹病毒Ⅱ型的鲫脾脏发生阳性反应。利用单抗4C2通过间接免疫荧光技术对鲤疱疹病毒Ⅱ型感染鎏金金鱼鳍条细胞系(RyuF-2)后ORF72蛋白表达情况进行检测,结果显示,感染后36 h可检测到阳性信号,并且随着感染时间的延长,细胞中ORF72的表达量明显增加。本试验制备的单克隆抗体4C2为体外进一步探究鲤疱疹病毒Ⅱ型在细胞内的复制机制及建立病鱼免疫检测手段奠定了基础。In order to establish an immune detection method based on antigen levels,the nucleocapsid protein ORF72 of cyprinid herpesvirusⅡ(CyHV-2)was prepared by prokaryotic expression method.The purified ORF72 protein was immunized to BALB/c mice,and the anti-ORF72 monoclonal antibodies designated as 1B3,4B1,4C2,5F4 and 5H9 were finally screened by hybridoma technology.Western blot analysis showed that the monoclonal antibodies specifically recognized recombinant protein ORF72 in vitro.The monoclonal antibody 4C2 detected the virus infection tissues of gibel crucian carp Carassius auratus gibelio by indirect immun of luorescence assay(IFA)only.IFA of the CyHV-2 replication on RyuF-2 detection with 4C2 showed that the positive signals were detected 36 hours after the virus infection,and the expression level of ORF72 protein was increased in the cell with time.Monoclonal antibody 4C2 contributes to research the replication mechanism of CyHV-2 in vitro and to establishment of the immune detection method for diseased fish.

关 键 词：异育银鲫 鲤疱疹病毒Ⅱ型 ORF72 单克隆抗体 间接免疫荧光 

分 类 号：S917.1[农业科学—水产科学]