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

J Virol Methods. 2007 Feb;139(2):175-80. doi: 10.1016/j.jviromet.2006.09.024. Epub 2006 Oct 31.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Animals
  • Antibodies, Viral / blood*
  • Baculoviridae / genetics
  • Baculoviridae / metabolism
  • Blotting, Western / methods*
  • Cell Line
  • Child, Preschool
  • Coronavirus Infections / diagnosis
  • Coronavirus Infections / immunology
  • Coronavirus OC43, Human / immunology
  • Coronavirus OC43, Human / isolation & purification*
  • Female
  • Gene Expression
  • Genetic Vectors
  • Humans
  • Male
  • Nucleocapsid Proteins / genetics
  • Nucleocapsid Proteins / immunology*
  • Recombinant Proteins / immunology*

Substances

  • Antibodies, Viral
  • Nucleocapsid Proteins
  • Recombinant Proteins
  • nucleocapsid protein, Coronavirus