Expression, Purification, and Application of SARS-CoV-2 Nucleocapsid Protein for Serological Detection of IgG and IgM Antibodies

Abstract

Background: SARS-CoV-2 is a novel coronavirus that has caused dramatic loss of life and poses an unprecedented public health challenge worldwide. The nucleocapsid (N) protein of SARS-CoV-2 is the most abundant viral protein and a potent immunogen.

Methods: In the current study, the N gene of SARS-CoV-2 was amplified from RNA extracted from a COVID-19 positive patient and then cloned into the pCold-I expression vector. The full-length His-tagged N protein was expressed in Escherichia coli (E. coli) using 0.5 mM IPTG and subsequently purified by nickel affinity chromatography. The purified N protein was characterized using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Furthermore, the purified N protein was applied in SARS-CoV-2 IgG and IgM ELISA immunoassays.

Results: The results showed that purification of the N protein in the presence of urea yielded a protein band of approximately 48 kDa on SDS-PAGE, corresponding to the full-length N protein. Additionally, Western blot analysis of the purified recombinant N protein showed a band of the same molecular weight. In the SARS-CoV-2 IgG ELISA assay, anti-N protein antibodies from a COVID-19 positive patient’s serum successfully recognized the coated N protein. In the IgM ELISA test, an N-HRP conjugate was used in ELISA wells to reveal the interaction of HRP-conjugated N protein with pre-coated anti-N protein antibodies (IgM isotype).

Conclusion: These results indicate that the expressed N protein of SARS-CoV-2 could serve as a valuable reagent for the development of antibody-based immunoassays to detect SARS-CoV-2 IgG and IgM antibodies.