Journal
JOURNAL OF VIROLOGICAL METHODS
Volume 139, Issue 2, Pages 175-180Publisher
ELSEVIER
DOI: 10.1016/j.jviromet.2006.09.024
Keywords
coronavirus; OC43 strain; nucleocapsid protein; baculovirus; insect cells; Western blot
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The nucleocapsid (N) gene of human coronavirus strain OC43 (HCoV-OC43) was amplified by reverse transcriptase-polymerase chain reaction, and cloned in pENTR (TM)/D-TOPO (R) plasmid. This plasmid containing the N gene was recombined with in a BaculoDirect (TM) baculovirus DNA designed in order to express N protein in fusion with a C-terminal polyhistidine tag containing V5 epitope. Sf21 cells were transfected with recombinant baculovirus DNA. Recombinant N protein was extracted from infected cells, analysed by SDS-PAGE and Western blot, and purified by Ni2+ affinity procedure. Sera from 100 healthcare workers and five 2-3-year-old children were tested in a Western blot assay using the purified recombinant N protein. All of the sera from adults and two of the sera from children have a positive result. (c) 2006 Elsevier B.V. All rights reserved.
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