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

Abstract

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.

Development and characterization of monoclonal antibodies specific for cyprinid herpesvirus 2

“…On the other hand, it can also be used in clarifying the process of virus invasion, and provide help for antiviral treatment (Zhu et al, 2020). At present, some anti‐CyHV‐2 monoclonal antibodies have been established, but they are mainly prepared by prokaryotic or eukaryotic expression virus proteins in vitro (Gao et al, 2022; Guo et al, 2022; Kong et al, 2017; Shen et al, 2018; Wu et al, 2020). Among them, proteins expressed in prokaryotic cells have simple conformation, lack the modifications and processing and often have some differences with the proteins in natural conditions.…”

“…Specific immunodiagnosis of CyHV-2 based Mabs are still necessary in elucidating the pathogenesis of CyHV-2. Several MAbs against recombinant proteins of CyHV-2 had been applied in detection and neutralization of CyHV-2 (Gao et al, 2022;Wu et al, 2020), but im-…”

“…The research of CyHV‐2 proteins focused on immune detection and protection. Mabs bound to recombinant capsid protein ORF72, ORF92 and ORF66 of CyHV‐2 and Mabs against expressed protein ORF121 can be effectively used in immunological detection of CyHV‐2 (Gao et al, 2022; Guo et al, 2022; Kong et al, 2017; Shen et al, 2018). Recombinant truncated membrane protein ORF25 was found to have certain immune protective effect against virus infection and was developed as a potential vaccine (Yuan et al, 2020; Zhou et al, 2015).…”

“…Specific immunodiagnosis of CyHV‐2 based Mabs are still necessary in elucidating the pathogenesis of CyHV‐2. Several MAbs against recombinant proteins of CyHV‐2 had been applied in detection and neutralization of CyHV‐2 (Gao et al, 2022; Wu et al, 2020), but immunodiagnosis and neutralization methods based on Mabs against intact CyHV‐2 particle are not yet been established. Moreover, identifying antigens with an immunoprotective effect is crucial for the development of vaccines.…”