Baculovirus expression of HCoV-OC43 nucleocapsid protein and development of a Western blot assay for detection of human antibodies against HCoV-OC43

Mourez, Thomas; Vabret, Astrid; Han, Yang; Dina, Julia; Legrand, Loïc; Corbet, Sandrine; Freymuth, François

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 Ni(2+) 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.