Production and application of monoclonal antibodies against ORF66 of cyprinid herpesvirus 2

Cyprinid herpesvirus 2(CyHV-2)is the main pathogen causing haematopoietic necrosis disease of goldfish (Carassius auratus auratus) and gibel carp (Carassius auratus gibelio), which has caused huge economic losses to aquaculture industry of goldfish and gibel carp around the world. Currently, various detection methods based on nucleic acids have been established for the detection of CyHV-2. However, there is still a lack of rapid and effective immunological detection technology. In this study, anti-CyHV-2 ORF66 monoclonal antibodies (MAbs) were prepared to use the recombinant ORF66 protein as the antigen. Firstly, the open reading frame of CyHV-2 ORF66 was cloned into the pET-28a vector and expressed in Escherichia coli. Three MAbs (2F11, 2G8, and 3D6) against recombinant ORF66 protein were developed by immunization of Balb/C mice. Among them, MAb-2F11 belonged to the IgG2b isotype, 2G8 and 3D6 belonged to the IgG1 isotype. Western blotting analysis was performed to assess the ability of the MAbs to bind to the ORF66 recombinant protein and CyHV-2 nucleocapsid protein ORF66. In addition, the MAb-2F11 was used to detect the virus particles that infected in cell line and tissues of gibel carp virus infection by immunological methods. These results indicated that the anti-CyHV-2 ORF66 MAb-2F11 prepared in this study could not only detect the presence of the virus but also provide a research tool for further studying the role of ORF66 in the process of CyHV-2 infection.