Baculovirus expression and purification of nucleocapsid protein of canine distemper virus for its evaluation as a diagnostic antigen.

Abstract

Canine distemper is a highly contagious disease of domestic dogs and various species of wild animals, and is associated with high mortality. It is caused by canine morbillivirus (formerly known as CDV or canine distemper virus), a member of the Morbillivirus genus under Paramyxoviridae family. The disease is endemic in many parts of the world, including India. For effective control of the disease, reliable diagnostic tools are of utmost importance. The circulating CDV strains in India belong to a novel "India1/Asia-5" lineage, highlighting the need for the development of new diagnostic tools. The nucleocapsid (N) protein is the major structural protein and one of the most immunogenic proteins of the virus; thus, it has been the preferred diagnostic target. Therefore, the present study was aimed at expressing the nucleocapsid protein of CDV using the baculovirus expression system in insect cells and its successful purification using affinity column chromatography. Expression and specificity of the recombinant N protein were confirmed using SDS-PAGE and Western blot analysis, revealing a band of approximately 58 kDa. The purified protein also reacted with CDV polyclonal serum, suggesting that the N protein was expressed in an immunoreactive form. Among the panel of monoclonal antibodies generated against CDV, only CDV-2F8 exhibited specific reactivity with the recombinant N protein, indicating the preservation of at least one linear epitope. The diagnostic suitability of the expressed protein was further confirmed by using it as a coating antigen in an indirect ELISA, which clearly distinguished between CDV-positive and negative dog serum samples. The recombinant N protein developed in the present study can facilitate large-scale field application of recombinant protein-based diagnostics for CDV, as it eliminates the need for live virus antigen in routine serodiagnostic assays.