Generation and application of a monoclonal antibody specific for the ORF121 of cyprinid herpesvirus 2

Cyprinid herpesvirus 2 (CyHV-2) is a viral pathogen worldwide and causing high mortality on goldfish and silver crucian carp (Carassius auratus gibelio). In order to establish a stable and sensitive immunological diagnostic approach, the recombinant ORF121 protein encoded by the CyHV-2 ORF121 gene, was selected as a capture antigen to identify cells and tissues infected with CyHV-2 by immunological methods in this study. Firstly, the open reading frame of CyHV-2 ORF121 was cloned into the PGEX-4T-3 vector and expressed in Escherichia coli. Purified recombinant ORF121 protein was then used as an antigen to prepare monoclonal antibodies, and an efficient hybridoma cell line was selected by dot-blot assay. The resulting mAb-3D9 was applied to detect CyHV-2 in infected caudal fin of Carassius auratus gibelio (GiCF) cells and fish tissues by western blotting, immunofluorescence assays and immunohistological asays. The monoclonal antibody could specifically identify CyHV-2 in infected GiCF cells and the gills, the kidney and the spleen tissues, and it could attenuate CPE by CyHV-2 in vitro, suggesting it can be applied for CyHV-2 detection in the crucian carp and ORF121 may be a candidate vaccine against CyHV-2.