Preparation and characterization of a mouse polyclonal antibody against the truncated ORFV113 recombinant protein of Orf virus

Orf is a contagious zoonotic disease caused by Orf virus (ORFV), posing a threat to both animal and human health. The ORFV113 gene, located in the terminal variable region of the ORFV genome, has been demonstrated as a significant virulence gene, but its function remains largely unknown. In the study, we first amplified the truncated version of the ORFV113 gene (ORFV113t) by removing its transmembrane domain at the 5′ end. We then constructed the pET-32a-ORFV113t recombinant plasmid and expressed the truncated ORFV113 recombinant protein in Escherichia coli (E.coli). The purified ORFV113t fusion protein was used to immunize mice and generate a polyclonal antibody. This polyclonal antibody was subsequently used to detect the expression and subcellular localization of the ORFV113 protein. Additionally, virus neutralization test was utilized to determine the neutralizing titer of the polyclonal antibody. The results demonstrated that we successfully expressed the ORFV113t recombinant protein in a prokaryotic expression system and generated a mouse-derived polyclonal antibody targeting the ORFV113t recombinant protein with a titer of 1:204,800. This antibody exhibited specificity for detecting the ORFV113 protein expressed in both prokaryotic and eukaryotic cells. The ORFV113 protein was found to be localized in the cytoplasm of infected Lamb testis (LT) cells. Notably, the polyclonal antibody demonstrated neutralizing activity against ORFV in vitro, with a neutralizing titer of 1:32. The prepared mouse anti-ORFV113t protein polyclonal antibody can be utilized for further study on potential functions of the ORFV113 protein in viral pathogenesis.