ABSTRACT
Objective To express and purify the recombinant nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and prepare antiserum from immunized mice. Methods The prokaryotic plasmid pET28a-N containing SARS-CoV-2 N gene was transformed into Escherichia coli BL21 (DE3). The expression of recombinant SARS-CoV-2 N protein was induced by isopropyl-β-D-thiogalactopyranoside. The Ni-NTA affinity chromatography column was used to purify the recombinant SARS-CoV-2 N protein, and antiserum was obtained from the BALB/c mice immunized with recombinant SARS-CoV-2 N protein combined with manganese adjuvant through intramuscular and subcutaneous injections. The reactions of recombinant SARS-CoV-2 N protein with SARS-CoV-2 N monoclonal antibodies and severe acute respiratory syndrome coronavirus (SARS-CoV) N polyclonal antibodies were detected by Western blotting. The reaction of mouse antiserum with the recombinant SARS-CoV-2 N protein expressed in the cells transfected with eukaryotic expression plasmid was examined by indirect immunofluorescence assay. Results The recombinant SARS-CoV-2 N protein was successfully induced and expressed as a soluble protein with a molecular weight of about 55 000. High concentration of purified protein was obtained. The results of Western blotting showed that the recombinant SARS-CoV-2 N protein could be specifically recognized by the SARS-CoV-2 N monoclonal antibodies and the SARS-CoV N polyclonal antibodies. The prepared mouse antiserum could also correctly recognize the recombinant SARS-CoV-2 N protein expressed in mammalian cells by indirect immunofluorescence assay. Conclusion Recombinant SARS-CoV-2 N protein has been successfully expressed and purified from the prokaryotic expression system, and mouse antiserum has been prepared, which lays a foundation for establishing a rapid SARS-CoV-2 diagnostic tool and further studying the function of SARS-CoV-2 N protein..
ABSTRACT
Objective To investigate the efficacy of neutralizing antibodies induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor-binding domain (RBD) and spike (S) protein S1 subunit. Methods The SARS-CoV-2 RBD and mouse immunoglobulin G1 (IgG1) Fc fragment (mFc) fusion protein expression plasmid pVRCRBD- mFc was constructed and transfected into human embryonic kidney 293T cells. The RBD-mFc fusion protein in the cell supernatants was detected by Western blotting. The effect of RBD-mFc in cell supernatants and CHO recombinant S1-human IgG1 Fc (S1-hFc) fusion protein on SARS-CoV-2 infection was detected by microneutralization test. BALB/c mice were immunized with plasmid pVRC-RBD-mFc and S1-hFc fusion protein via intramuscular injection. Anti-S1 IgG antibodies in mouse sera were detected by enzyme-linked immunosorbent assay (ELISA), and the virus neutralization activity of mouse sera was detected by microneutralization test. Results The RBD-mFc fusion protein could be detected in the culture supernatants of 293T cells transfected with the plasmid pVRC-RBD-mFc, the concentrated supernatants and the S1- hFc fusion protein could inhibit SARS-CoV-2 infection on Vero E6 cells in a concentration-dependent manner. Anti-S1 IgG antibodies could be detected in the sera of mice immunized with plasmid pVRC-RBD-mFc and S1-hFc fusion protein, and the sera of both groups could neutralize SARS-CoV-2 infection. The serum antibody titers and virus neutralization activity of S1- hFc fusion protein immunized mice were significantly higher than those of plasmid pVRC-RBD-mFc immunized mice (both P<0.01). Conclusion Both SARS-CoV-2 RBD and S1 subunit may be used as effective vaccine antigens. Compared with DNA vaccine, recombinant subunit vaccine can induce neutralizing antibody more effectively..