Identification of major immunogenic proteins from Cyprinid herpesvirus 2

In order to obtain promising tools for improved diagnostics and new vaccine development, we purified Cyprinid herpesvirus 2 (CyHV-2) particles and identified more immunologic proteins in CyHV-2 particles by LC-MS. Firstly, the caudal fin of Carassius auratus gibelio (GiCF) cell line was infected with CyHV-2, and viral particles were purified from the cell supernatant by sucrose density gradient in combination with ultracentrifugation. The purified virions were observed by transmission electron microscope. Then the prepared purified virions were used to immunize mice to prepare an anti-CyHV-2 polyclonal antibody, and at the same time purified virions were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and stained with coomassie brilliant blue, and then subjected to Western blotting analysis and mass spectrometry identification using anti-CyHV-2 polyclonal antibody. Transmission electron microscope (TEM) results showed that a large amount of intact CyHV-2 virus particles were detected at 50%-66% sucrose density and virus particles without capsule were occasionally observed. Western blotting results showed that some protein bands had specific immunoreactivity with anti-CyHV-2 polyclonal antibody. 8 major immunogenic proteins were further identified as ORF92, ORF115, ORF25, ORF57, ORF66, ORF72, ORF131and ORF132 through mass spectrometry identification. Among them, ORF92, ORF66 and ORF72 are capsid proteins, and ORF115, ORF25, ORF131 and ORF132 are membrane proteins. The capsid protein and membrane protein of the virus play an important role in virus infection. In conclusion, the major immunogenic proteins of CyHV-2 virion were identified. This was fundamental and applied research on CyHV-2. Indeed, this knowledge is crucial for understanding the biology and pathogenesis of CyHV-2 infection and for facilitating the development of efficacious protein-based diagnostic methods and vaccine candidates.