Detection of grass carp reovirus (GCRV) with monoclonal antibodies
Detection of grass carp reovirus (GCRV) with monoclonal antibodies
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DOI：
10.1007/s00705-013-1864-7
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发表时间：
2014-04-01
影响因子：
2.7
通讯作者：
Lin Xiangmei
中科院分区：
文献类型：
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作者：
Jing Hongli;Zhang Lifeng;Lin Xiangmei
草鱼呼肠孤病毒（GrasscarpReovirus，GCRV）是引起草鱼出血病的病原。该病是鲤鱼最严重的传染病，造成草鱼和青鱼鱼种的严重损失。本研究构建了带有组氨酸标签的重组病毒核心蛋白VP 4，并在大肠杆菌中高效表达。大肠杆菌中表达的蛋白主要以包涵体形式存在。表达的VP 4蛋白能被抗His标签单克隆抗体和山羊抗GCRV血清识别。制备了4株抗重组VP 4蛋白的单克隆抗体（16 B7、39 E12、13 C3和14 D1）。这些单克隆抗体在ELISA试验中不与除GCRV外的任何测试病毒或鱼细胞系反应。在蛋白质印迹分析中，当使用GCRV的重组VP 4蛋白作为抗原时观察到蛋白质条带，但是当使用GCRV的天然衣壳蛋白作为抗原时观察到68-kDa条带。建立了检测GCRV的双抗体夹心ELISA方法。检测限为10（5）TCID 50/mL GCRV。
Grass carp reovirus (GCRV) is a pathogen that causes hemorrhagic disease of grass carp. It is the most serious infectious disease of carp and causes serious losses of fingerlings of grass carp and black carp. In this study, a recombinant VP4, one of the viral core proteins, was constructed with a histidine tag and expressed at a high level in E. coli, and the expressed protein was mainly found in the form of inclusion bodies. The expressed VP4 protein was recognized by an anti-His-tag monoclonal antibody and goat anti-GCRV serum. Four monoclonal antibodies (16B7, 39E12, 13C3 and 14D1) against the recombinant VP4 protein were produced. These MAbs did not react with any of the tested viruses or fish cells lines in the ELISA tests except GCRV. In western blotting analysis, a protein band was observed when the recombinant VP4 protein of GCRV was used as an antigen, but a 68-kDa band was observed when natural capsid proteins of GCRV were used as antigens. Furthermore, a sandwich ELISA was developed for detection of GCRV. The detection limit of the test was 10(5) TCID50 of GCRV per mL.