title: Expression of recombinant West Nile virus prM protein fused to an affinity tag for use as a diagnostic antigen.
abstract: Previous studies have concluded that the Flavivirus prM protein is a suitable viral antigen to distinguish serologically between infections with closely related Flaviviruses (Cardosa et al., 2002). To express the recombinant West Nile virus (WNV) prM antigen fused to a suitable affinity tag for purification, a series of prM-His-tag and prM-V5-tag fusion proteins were generated. Analysis of the prM-His-tag fusion proteins revealed that either prM epitopes were disrupted or the His-tag was not presented properly depending on the location of the His tag and the presence of the prM transmembrane domains in these constructs. This identified domains critical for proper folding of prM, and arrangements that allowed the correct presentation of the His-tag. However, the inclusion of the V5 epitope tag fused to the C terminus of prM allowed formation of the authentic antigenic structure of prM and the proper presentation of the V5 epitope. Capture of tagged recombinant WNV(NY99) prM antigen to the solid phase with anti-V5 antibody in ELISA enabled the detection of prM-specific antibodies in WNV(NY99)-immune horse serum, confirming its potential as a useful diagnostic reagent.
authors.affiliation: Australian Infectious Diseases Research Centre, School of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia 4072, Queensland, Australia.
authors.forename: Y X
authors.initials: YX
authors.lastname: Setoh
authors.forename: J
authors.initials: J
authors.lastname: Hobson-Peters
authors.forename: N A
authors.initials: NA
authors.lastname: Prow
authors.forename: P R
authors.initials: PR
authors.lastname: Young
authors.forename: R A
authors.initials: RA
authors.lastname: Hall
mesh_terms: D000330:Aedes; D000345:Affinity Labels / Q000032:analysis*; D000818:Animals; D000914:Antibodies, Viral / Q000276:immunology; D000956:Antigens, Viral / Q000276:immunology; D002460:Cell Line; D002522:Chlorocebus aethiops; D004797:Enzyme-Linked Immunosorbent Assay; D006736:Horses / Q000276:immunology; D007106:Immune Sera; D011993:Recombinant Fusion Proteins / Q000276:immunology; D014759:Viral Envelope Proteins / Q000235:genetics / Q000276:immunology*; D014764:Viral Proteins / Q000276:immunology; D015678:Viral Structural Proteins / Q000276:immunology; D014901:West Nile Fever / Q000175:diagnosis / Q000276:immunology*; D014902:West Nile virus / Q000276:immunology*
chemical_list: D000345:Affinity Labels; D000914:Antibodies, Viral; D000956:Antigens, Viral; D007106:Immune Sera; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; D014764:Viral Proteins; D015678:Viral Structural Proteins; C074932:prM protein, Flavivirus