ABSTRACT
<p><b>OBJECTIVE</b>To clone and express nucleocapsid (N) protein of the severe acute respiratory syndrome (SARS)-associated coronavirus, and to evaluate its antigenicity and application value in the development of serological diagnostic test for SARS.</p><p><b>METHODS</b>SARS-associated coronavirus N protein gene was amplified from its genomic RNA by reverse transcript nested polymerase chain reaction (RT-nested-PCR) and cloned into pBAD/Thio-TOPO prokaryotic expression vector. The recombinant N fusion protein was expressed and purified, and its antigenicity and specificity was analyzed by Western Blot, to establish the recombinant N protein-based ELISA for detection of IgG antibodies to SARS-associated coronavirus, and SARS-associated coronavirus lysates-based ELISA was compared parallelly.</p><p><b>RESULTS</b>The recombinant expression vector produced high level of the N fusion protein after induction, and that protein was purified successfully by affinity chromatography and displayed higher antigenicity and specificity as compared with whole virus lysates.</p><p><b>CONCLUSION</b>The recombinant SARS-associated coronavirus N protein possessed better antigenicity and specificity and could be employed to establish a new, sensitive, and specific ELISA for SARS diagnosis.</p>
Subject(s)
Humans , Antibodies, Viral , Blood , Blotting, Western , Cloning, Molecular , Enzyme-Linked Immunosorbent Assay , Gene Expression , Genome, Viral , Immunoglobulin G , Blood , Nucleocapsid Proteins , Genetics , Allergy and Immunology , Metabolism , RNA, Viral , Genetics , Recombinant Proteins , Metabolism , Reverse Transcriptase Polymerase Chain Reaction , Severe acute respiratory syndrome-related coronavirus , Genetics , Allergy and Immunology , Metabolism , Severe Acute Respiratory Syndrome , Blood , Diagnosis , VirologyABSTRACT
<p><b>OBJECTIVE</b>Angiotensin-converting enzyme 2 (ACE-2) has been identified as a functional receptor of severe acute respiratory syndrome coronavirus (SARS-CoV), so its gene was cloned and eukaryotic expressed for further insight into mechanisms in SARS-CoV entry and pathogenesis, as well as development of a safe and reliable neutralization assay for SARS-CoV.</p><p><b>METHODS</b>Total RNA was extracted from right atrial tissue of a patient with right heart failure resected during a valvular replacement surgery by Trizol one-step method, and the full-length ACE-2 encoding gene was acquired by RT-nested-PCR. The ACE-2 encoding gene was then cloned into pcDNA4/HisMax-TOPO eukaryotic expression vector to construct the recombinant plasmid pcDNA4/ ACE-2, which was then transfected into 293 T cell and ACE-2 eukaryotic transient expression was detected by Western Blot. Syncytia inhibition assay was established to detect SARS-CoV neutralizing antibody, and compared parallelly with SARS pseudovirus neutralization assay.</p><p><b>RESULTS</b>The recombinant plasmid pcDNA4/ ACE-2 could express ACE-2 protein in eukaryotic cells and induce cell-cell fusion between S protein- and ACE2-expressing cells. This cell-cell fusion assay could be used to detect SARS-CoV neutralizing antibody.</p><p><b>CONCLUSION</b>SARS-CoV receptor ACE-2 gene was successfully cloned and eukaryotic expressed, and used to establish syncytia inhibition assay for SARS-CoV neutralizing antibody assay.</p>