Baculovirus expression of HCoV-OC43 nucleocapsid protein and development of a Western blot assay for detection of human antibodies against HCoV-OC43.
The nucleocapsid (N) gene of human coronavirus strain OC43 (HCoV-OC43) was amplified by reverse transcriptase-polymerase chain reaction, and cloned in pENTR/D-TOPO plasmid. This plasmid containing the N gene was recombined with in a BaculoDirect 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.
Mourez
Thomas
T
Laboratory of Human and Molecular Virology, University Hospital of Caen, Avenue Georges Clemenceau, 14 033 Caen Cedex, France.
Vabret
Astrid
A
Han
Yang
Y
Dina
Julia
J
Legrand
Loïc
L
Corbet
Sandrine
S
Freymuth
François
F
D000328:Adult; D000818:Animals; D000914:Antibodies, Viral / Q000097:blood*; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism; D015153:Blotting, Western / Q000379:methods*; D002460:Cell Line; D002675:Child, Preschool; D018352:Coronavirus Infections / Q000175:diagnosis / Q000276:immunology; D000086462:Coronavirus Nucleocapsid Proteins; D028962:Coronavirus OC43, Human / Q000276:immunology / Q000302:isolation & purification*; D005260:Female; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D008297:Male; D019590:Nucleocapsid Proteins / Q000235:genetics / Q000276:immunology*; D011994:Recombinant Proteins / Q000276:immunology*
D000914:Antibodies, Viral; D000086462:Coronavirus Nucleocapsid Proteins; D019590:Nucleocapsid Proteins; D011994:Recombinant Proteins