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<p>Abstract</p> <p>Background</p> <p>The duck plague virus (DPV) UL46 protein (VP11/12) is a 739-amino acid tegument protein encoded by the <it>UL46 </it>gene. We analyzed the amino acid sequence of UL46 using bioinformatics tools and defined the main antigenic domains to be between nucleotides 700-2,220 in the <it>UL46 </it>sequence. This region was designated UL46M. The DPV <it>UL46 </it>and <it>UL46M </it>genes were both expressed in <it>Escherichia coli </it>Rosetta (DE3) induced by isopropy1-β-<smcaps>D</smcaps>-thiogalactopyranoside (IPTG) following polymerase chain reaction (PCR) amplification and subcloning into the prokaryotic expression vector pET32a(+). The recombinant proteins were purified using a Ni-NTA spin column and used to generate the polyclonal antibody against UL46 and UL46M in New Zealand white rabbits. The titer was then tested using enzyme-linked immunosorbent assay (ELISA) and agar diffusion reaction, and the specificity was tested by western blot analysis. Subsequently, we established Dot-ELISA using the polyclonal antibody and applied it to DPV detection.</p> <p>Results</p> <p>In our study, the DPV UL46M fusion protein, with a relative molecular mass of 79 kDa, was expressed in <it>E. coli </it>Rosetta (DE3). Expression of the full <it>UL46 </it>gene failed, which was consistent with the results from the bioinformatic analysis. The expressed product was directly purified using Ni-NTA spin column to prepare the polyclonal antibody against UL46M. The titer of the anti-UL46M antisera was over 1:819,200 as determined by ELISA and 1:8 by agar diffusion reaction. Dot-ELISA was used to detect DPV using a 1:60 dilution of anti-UL46M IgG and a 1:5,000 dilution of horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG.</p> <p>Conclusions</p> <p>The anti-UL46M polyclonal antibody reported here specifically identifies DPV, and therefore, it is a promising diagnostic tool for DPV detection in animals. UL46M and the anti-UL46M antibody can be used for further clinical examination and research of DPV.</p>