Kong S Y, Jiang Y S, Wang Q, Lu J F, Xu D, Lu L Q
National Pathogen Collection Center for Aquatic Animals, Key Laboratory of Aquatic Genetic Resources of Ministry of Aquaculture, Shanghai Ocean University, Shanghai, China.
Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou, China.
J Fish Dis. 2017 Dec;40(12):1791-1798. doi: 10.1111/jfd.12648. Epub 2017 May 26.
Cyprinid herpesvirus 2 (CyHV-2) is the main pathogen responsible for causing haematopoietic necrosis disease in Carassius auratus gibelio. Although many nucleic acid-based diagnostic methods have been applied, no stable and sensitive immunological diagnostic approaches have been reported. In this study, to detect CyHV-2 in clinical samples using immunological methods, recombinant ORF72 protein (pORF72), encoded by the CyHV-2 ORF72 gene, was used as a capture antigen to identify blood and tissues infected with CyHV-2. First, ORF72 gene was amplified from the CyHV-2 genome and cloned into a PGEX-4t-3 expression vector to produce pORF72 in Escherichia coli. The purified pORF72 was used as an immunogen to prepare monoclonal antibodies. The Western blotting assays revealed that the monoclonal antibody could specifically identify the pORF72. Furthermore, an immunohistochemical protocol and a blood smear method were established to detect CyHV-2 in carps. The results indicate that the monoclonal antibody against pORF72 could be utilized as an effective detection tool for haematopoietic necrosis disease in Carassius auratus gibelio.
鲤疱疹病毒2型（CyHV-2）是导致异育银鲫造血坏死病的主要病原体。尽管已经应用了许多基于核酸的诊断方法，但尚未报道稳定且灵敏的免疫诊断方法。在本研究中，为了使用免疫方法检测临床样本中的CyHV-2，将由CyHV-2 ORF72基因编码的重组ORF72蛋白（pORF72）用作捕获抗原，以鉴定感染CyHV-2的血液和组织。首先，从CyHV-2基因组中扩增出ORF72基因，并将其克隆到PGEX-4t-3表达载体中，以便在大肠杆菌中产生pORF72。纯化后的pORF72用作免疫原制备单克隆抗体。蛋白质免疫印迹分析表明，该单克隆抗体能够特异性识别pORF72。此外，还建立了免疫组织化学方法和血涂片方法来检测鲤鱼中的CyHV-2。结果表明，针对pORF72的单克隆抗体可作为检测异育银鲫造血坏死病的有效工具。
