Abstract: 【Objective】 A prokaryotic expression system for the nucleocapsid（N） protein of porcine deltacoronavirus（PDCoV） was constructed, and polyclonal antibodies were prepared to provide reference for the development of novel vaccines, diagnostic reagents, and in-depth research into the pathogenic mechanisms of PDCoV. 【Method】 RNA from the PDCoV-HeN17 strain was extracted and amplified by RT-PCR, and the N gene was cloned into the pGEX-6P-1 prokaryotic expression vector to construct a recombinant prokaryotic expression plasmid. This plasmid was transformed into Escherichia coli BL21（DE3） competent cells, and expression was induced with IPTG. The recombinant protein was purified using Glutathione Sepharose 4B affinity chromatography, and the tag was removed with 3C protease. Polyclonal antibodies were prepared using the purified N protein as the immunogen to immunize 3-month-old New Zealand white rabbits.The antibody titer was determined by indirect enzyme-linked immunosorbent assay（ELISA）, and the antibodies were characterized by Western blotting, indirect immunofluorescence（IFA） and immunoprecipitation（IP） assays. 【Result】 The prokaryotic expression vector pGEX-6p-1-PDCoV-N was successfully constructed, and the obtained PDCoV-N-GST fusion protein was primarily expressed in a soluble form, with an approximate size of 69 kD. After the GST tag was removed, a single band of the target protein was obtained, with a concentration determined to be 12.6 mg/mL. The produced PDCoV-N protein could react with PDCoV positive serum to produce a single specific band. The prepared polyclonal antibody against PDCoV-N from rabbits reached a titer of 1∶4096000 and could specifically recognize the Flag-N protein expressed in HEK 293T cells transfected with pCAGGS-Flag-N and the PDCoV-N protein expressed in LLC-PK1cells infected with PDCoV. It could also detect the PDCoV-N protein expressed in HEK 293T cells transfected with the eukaryotic expression vector pCAGGS-Flag-N. 【Conclusion】 An efficient prokaryotic expression system for PDCoV-N is successfully constructed, yielding PDCoV-N protein with high purity, concentration and good immunogenicity. The prepared anti-PDCoV-N polyclonal antibodies from rabbits exhibited high titers, strong affinity, and specificity, making them suitable for applications in Western blotting, IFA, and IP assays.