Cloning, sequencing, expression, and purification of SARS-associated coronavirus nucleocapsid protein for serodiagnosis of SARS

Abstract

A novel coronavirus has been associated with a worldwide outbreak of atypical pneumonia referred to as Severe Acute Respiratory Syndrome (SARS-CoV). SARS-CoV nucleocapsid (N) protein has been cloned sequenced and expressed in Escherichia coli strain. Purified N protein was used to measure the SARS-CoV specific IgG antibodies from 16 SARS-CoV infected patients' sera and from 131 control subjects using ELISA assay. Specific antibody responses to the purified recombinant N protein after 10, 20, and 30 days of disease onset were observed in 13 of 16 (81.3%), 16 of 16 (100%) and 16 of 16 (100%) SARS patients sera, respectively. Comparison of detection results with a commercially available diagnostic kit coated with a mixture of SARS-CoV viral proteins showed 9 of 16 (56.3%), 13 of 16 (81.3%), and 15 of 16 (93.7%) positive responses, respectively. None of 131 control sera gave positive reaction in either assay. This data suggests that the N protein of SARS-CoV is immunodominant and this ELISA based test assay for detecting the SARS-CoV N antigen may hold a significant value for SARS diagnosis.

Analysis of preferred codon usage in the coronavirus N genes and their implications for genome evolution and vaccine design

“…In an interesting study, the N gene of the CoV was found to be more effective for evaluating the codon usage bias than the S gene (Ahn et al, 2009). Studies reported that the N protein produced from prokaryotes has been used to generate specific antibodies against various animal coronaviruses including SARS (Loa et al, 2004;Timani et al, 2004;Wu et al, 2004aWu et al, , 2004bBlanchard et al, 2011). The recombinant antigenic N protein from hCoV OC43 used against the rabbit polyclonal antibodies specific for hCoV OC43 and did not crossreact with other coronaviruses (SARS CoV and hCoV 229E) (Liang et al, 2013).…”

Use of the COOH Portion of the Nucleocapsid Protein in an Antigen-Capturing Enzyme-Linked Immunosorbent Assay for Specific and Sensitive Detection of Severe Acute Respiratory Syndrome Coronavirus

“…Since the specific antibodies to nucleocapsid (N) protein are most abundant in the sera from SARS patients (5), several groups have developed some recombinant N protein-based ELISAs. In general, these new assays are more specific and sensitive than the ELISA based on whole-virus lysate, but some false-positive results still occurred with sera from non-SARS patients or healthy people, even with sera collected several years before the SARS outbreak (3,7,8,(10)(11)(12).Since the N proteins of the known coronaviruses are relatively conserved, the expressed N protein of the SARS-CoV in Escherichia coli cross-reacts with polyclonal antisera of some known animal coronaviruses in antigenic group I, including transmissible gastroenteritis virus, feline infectious peritonitis virus, and canine coronavirus (9), which raised concerns of potential false positives when the recombinant N protein of the SARS-CoV, whole-virus antigen extracts, or virus-infected cells are used as reagents. However, this concern is very minimal, since the two known human coronaviruses (strains 229E and OC43) do not cause severe clinical diseases and we still do not have data about the prevalence of the antibodies to other coronaviruses or relevant microorganisms in human populations (4,6,8).…”

“…Since the specific antibodies to nucleocapsid (N) protein are most abundant in the sera from SARS patients (5), several groups have developed some recombinant N protein-based ELISAs. In general, these new assays are more specific and sensitive than the ELISA based on whole-virus lysate, but some false-positive results still occurred with sera from non-SARS patients or healthy people, even with sera collected several years before the SARS outbreak (3,7,8,(10)(11)(12).…”

Immunoreactivity characterisation of the three structural regions of the human coronavirus OC43 nucleocapsid protein by Western blot: Implications for the diagnosis of coronavirus infection

“…Prokaryotic-expressed NPs have been used successfully as antigens in ELISA-based assays for the detection of antibodies specific to many viruses, including SARS-CoV and several animal coronaviruses (Blanchard et al, 2011;Loa et al, 2004;Timani et al, 2004;Wu et al, 2004). In the current study, large quantities of soluble recombinant HCoV-OC43 NP from E. coli were expressed and purified in the presence of 0.1% CHAPS.…”