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Objective To identify key amino acid variations of major proteins from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by biophysical methods. Methods Through amino acid sequence alignment, classification of variant amino acid residues, three-dimensional structure reconstruction of proteins, and electrostatic interaction analysis of amino acid residues, the key amino acid variations of major proteins from SARS-CoV-2 was analyzed with RaTG13, the bat coronavirus with the highest homology, as the reference. Results At least ten amino acid variations that affect the possible electrostatic interactions were identified in RNA-dependent RNA polymerase (RdRp), exoribonuclease (ExoN), uridylate-specific endoribonuclease (NendoU), and spike (S) protein from SARS-CoV-2. These variations may affect the spatial conformation and biological functions of the proteins. Conclusion The key amino acid variations of the major proteins from SARS-CoV-2 have been preliminarily identified, providing clues for understanding the genetic, pathogenic and epidemiological characteristics of the virus..
RESUMO
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..