Preparation and characterization of Cyprinid herpesvirus 3 interleukin 10 monoclonal antibody

Abstract:

Cyprinid herpesvirus 3 （CyHV3）， also known as Koi herpesvirus （KHV）， is a highly contagious pathogen that threatens the sustainability of cyprinid aquaculture. The ORF134 gene of CyHV3 encodes an interleukin （IL） 10 like protein. In this study， we constructed a prokaryotic expression plasmid of ORF134 and transformed it into the E. coli Rosetta （DE3） cells for expression of recombinant protein. The recombinant CyHV3-IL10 protein was purified by size exclusion chromatography and used for immunization of mice to generate monoclonal antibodies. The antibodies were then characterized by ELISA， Western blotting and immunofluorescent assay. SDS-PAGE analysis showed that the recombinant CyHV3-IL10 protein was approximately 18 ku in size and was expressed mainly in the form of inclusion bodies. The inclusion bodies were denatured， refolded and subjected to size exclusion chromatography. The recombinant CyHV3-IL10 protein was of high purity. Western blotting and immunofluorescence analyses revealed that the CyHV3-IL10 monoclonal antibodies recognized the recombinant CyHV3-IL10 protein expressed in the E. coli and HEK293 cells and could specifically detect the HEK293 cells expressing the CyHV3-IL10 protein. In summary， the availability of CyHV3-IL10 monoclonal antibodies lays the foundation for the functional study of CyHV3-IL10 and the establishment of CyHV3 diagnostic tools， and provides novel strategies for the prevention and control of the diseases caused by CyHV3.