Objective:To realize prokaryotic expression, purification and identification of 2019-novel Coronavirus (2019-nCoV) nucleocapsid protein (NP), and apply it to the serological diagnosis.Methods:The synthetic 2019-nCoV NP gene was cloned into the prokaryotic expression vector pET28a to construct expression plasmid, and then purified by Ni-chelating affinity. SDS-polyacrylamide gel electrophoresis (SDS-PAGE), indirect enzyme-linked immunosorbent assay (ELISA), Western blot (WB), and immunochromatography were used to test the purified protein. Indirect ELISA reaction conditions were optimized for serum antibody detection.Results:The relative molecular mass of recombinant NP was about 50×10 3 after SDS-PAGE electrophoresis, which was consistent with the expectation. Indirect ELISA and WB results showed that it could specifically bind to the serum of patients infected with 2019-nCoV. The detection limit of NP was 0.2 ng/ml by immunochromatography. The sera from 32 patients infected with 2019-nCoV and the control sera were detected by indirect ELISA, and the results showed that they were clearly clustered. Conclusions:Prokaryotic expression of 2019-nCoV NP has good immunogenicity and can be used for the development of serological diagnostic reagents.