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. 2004 Aug;30(4):309-12.
doi: 10.1016/j.jcv.2004.01.001.

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

Khalid Amine Timani  1 , Li Ye, Linbai Ye, Ying Zhu, Zhenghui Wu, Zuojiong Gong
Affiliations

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

Khalid Amine Timani et al. J Clin Virol. 2004 Aug.

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.

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Figures

Fig. 1
Fig. 1
SDS-PAGE analysis of the expression of the nucleocapsid protein in E. coli. Lanes 1 and 2: crude material from bacterial cultures containing the pETHis expression vectors with cDNA insert encoding the N protein, before (lane 2) and after (lane 1) the 2 h induction. Lane 3: purified recombinant protein by Ni-NTA affinity column. M: markers with their corresponding molecular masses.
See this image and copyright information in PMC

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