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.

Beijing has been one of the epicenters attacked most severely by the SARS-CoV (severe acute respiratory syndrome-associated coronavirus) since the first patient was diagnosed in one of the city's hospitals. We now report complete genome sequences of the BJ Group, including four isolates (Isolates BJ01, BJ02, BJ03, and BJ04) of the SARS-CoV. It is remarkable that all members of the BJ Group share a common haplotype, consisting of seven loci that differentiate the group from other isolates published to date. Among 42 substitutions uniquely identified from the BJ group, 32 are non-synonymous changes at the amino acid level. Rooted phylogenetic trees, proposed on the basis of haplotypes and other sequence variations of SARS-CoV isolates from Canada, USA, Singapore, and China, gave rise to different paradigms but positioned the BJ Group, together with the newly discovered GD01 (GD-Ins29) in the same clade, followed by the H-U Group (from Hong Kong to USA) and the H-T Group (from Hong Kong to Toronto), leaving the SP Group (Singapore) more distant. This result appears to suggest a possible transmission path from Guangdong to Beijing/Hong Kong, then to other countries and regions.
Genome, Viral ; Haplotypes ; Humans ; Mutation ; Open Reading Frames ; Phylogeny ; SARS Virus ; genetics