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1.
Int J Biol Macromol ; 200: 428-437, 2022 Mar 01.
Article in English | MEDLINE | ID: covidwho-1633983

ABSTRACT

Nucleocapsid protein (N protein) is the primary antigen of the virus for development of sensitive diagnostic assays of COVID-19. In this paper, we demonstrate the significant impact of dimerization of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) N-protein on sensitivity of enzyme-linked immunosorbent assay (ELISA) based diagnostics. The expressed purified protein from E. coli is composed of dimeric and monomeric forms, which have been further characterized using biophysical and immunological techniques. Indirect ELISA indicated elevated susceptibility of the dimeric form of the nucleocapsid protein for identification of protein-specific monoclonal antibody as compared to the monomeric form. This finding also confirmed with the modelled structure of monomeric and dimeric nucleocapsid protein via HHPred software and its solvent accessible surface area, which indicates higher stability and antigenicity of the dimeric type as compared to the monomeric form. The sensitivity and specificity of the ELISA at 95% CI are 99.0% (94.5-99.9) and 95.0% (83.0-99.4), respectively, for the highest purified dimeric form of the N protein. As a result, using the highest purified dimeric form will improve the sensitivity of the current nucleocapsid-dependent ELISA for COVID-19 diagnosis, and manufacturers should monitor and maintain the monomer-dimer composition for accurate and robust diagnostics.


Subject(s)
COVID-19 Testing/methods , Coronavirus Nucleocapsid Proteins/chemistry , Enzyme-Linked Immunosorbent Assay/methods , SARS-CoV-2/immunology , Antibodies, Viral/immunology , Circular Dichroism , Coronavirus Nucleocapsid Proteins/biosynthesis , Coronavirus Nucleocapsid Proteins/immunology , Coronavirus Nucleocapsid Proteins/isolation & purification , Dimerization , Epitopes/chemistry , Escherichia coli/genetics , Humans , Immunoglobulin G/immunology , Models, Molecular , Phosphoproteins/biosynthesis , Phosphoproteins/chemistry , Phosphoproteins/immunology , Phosphoproteins/isolation & purification , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/immunology , Recombinant Proteins/isolation & purification , Sensitivity and Specificity
2.
Protein Expr Purif ; 186: 105908, 2021 10.
Article in English | MEDLINE | ID: covidwho-1243167

ABSTRACT

The current standard for the diagnosis of COVID-19 is the nucleic acid test of SARS-CoV-2 RNA, however, virus antibody detection has the advantages of convenient sample collection, high throughout, and low cost. When combining detection with nucleic acid detection, antibody detection can effectively compensate for nucleic acid detection. Virus infection always induce high antibody titer against SARS-CoV-2 nucleocapsid protein (N protein), which can be used to detect COVID-19 at both infected and convalescent patients. In this study we reported the expression and purification of N protein in E.coli from inclusion bodies by a combination of two cation exchange chromatography, and the yield of N protein was around 50 mg/L fermentation broth with more than 90% purity. A corresponding colloidal gold detection kit prepared with our purified N protein was used to verify the efficiency and accuracy our N protein in antibody detection method. Of the 58 COVID-19 PCR positive patients' inactivated serum samples, 40 samples were IgM positive (69.0%), and 42 samples were IgG positive (72.4%), and all 95 COVID-19 negative patients' inactivated serum samples were both IgM and IgG negative. Our results indicates that the refolded soluble N protein could be used for the preliminary detection of IgG and IgM antibodies against SARS-CoV- 2.


Subject(s)
Antibodies, Viral/blood , COVID-19 Serological Testing/methods , Coronavirus Nucleocapsid Proteins/genetics , Coronavirus Nucleocapsid Proteins/immunology , SARS-CoV-2/immunology , Coronavirus Nucleocapsid Proteins/biosynthesis , Coronavirus Nucleocapsid Proteins/isolation & purification , Escherichia coli/genetics , Humans , Immunoglobulin G/blood , Immunoglobulin M/blood , Inclusion Bodies , Phosphoproteins/biosynthesis , Phosphoproteins/genetics , Phosphoproteins/immunology , Phosphoproteins/isolation & purification , Recombinant Proteins/biosynthesis , Recombinant Proteins/immunology , Recombinant Proteins/isolation & purification , SARS-CoV-2/genetics , Sensitivity and Specificity
3.
Science ; 371(6532): 926-931, 2021 02 26.
Article in English | MEDLINE | ID: covidwho-1048642

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral proteins interact with the eukaryotic translation machinery, and inhibitors of translation have potent antiviral effects. We found that the drug plitidepsin (aplidin), which has limited clinical approval, possesses antiviral activity (90% inhibitory concentration = 0.88 nM) that is more potent than remdesivir against SARS-CoV-2 in vitro by a factor of 27.5, with limited toxicity in cell culture. Through the use of a drug-resistant mutant, we show that the antiviral activity of plitidepsin against SARS-CoV-2 is mediated through inhibition of the known target eEF1A (eukaryotic translation elongation factor 1A). We demonstrate the in vivo efficacy of plitidepsin treatment in two mouse models of SARS-CoV-2 infection with a reduction of viral replication in the lungs by two orders of magnitude using prophylactic treatment. Our results indicate that plitidepsin is a promising therapeutic candidate for COVID-19.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , Depsipeptides/pharmacology , Peptide Elongation Factor 1/antagonists & inhibitors , SARS-CoV-2/drug effects , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/pharmacology , Adenosine Monophosphate/therapeutic use , Alanine/analogs & derivatives , Alanine/pharmacology , Alanine/therapeutic use , Animals , Antiviral Agents/therapeutic use , COVID-19/prevention & control , COVID-19/virology , Coronavirus Nucleocapsid Proteins/biosynthesis , Coronavirus Nucleocapsid Proteins/genetics , Depsipeptides/administration & dosage , Depsipeptides/therapeutic use , Drug Evaluation, Preclinical , Female , HEK293 Cells , Humans , Lung/virology , Mice, Inbred C57BL , Mutation , Peptides, Cyclic , Phosphoproteins/biosynthesis , Phosphoproteins/genetics , RNA, Viral/biosynthesis , RNA, Viral/genetics , SARS-CoV-2/genetics , SARS-CoV-2/physiology , Virus Replication/drug effects
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