title: Expression of recombinant Norwalk-like virus capsid proteins using a bacterial system and the development of its immunologic detection.
abstract: The capsid protein of Norwalk-like virus (NLV) isolates NLV-36 (Mexico virus type, genogroup II [GII]), NLV-21 (Lordsdale virus type, GII), NLV-114 (untyped GII virus), and NLV-96-908 (KY89 virus type, GI) have been expressed in an Escherichia coli system. The expressed recombinant NLV capsid proteins, fused with maltose binding protein (MBP-rV) and thioredoxin (TRX-rV) in E. coli lysate, were analyzed using sodium dodecyl sulfate-polyacrylamide gel eletrophoresis. Rabbit IgG (R-IgG) in hyperimmune serum has been raised against MBP-rV-36 capsid protein and was purified before further study. Detection of TRX-rVs using an enzyme-linked immunosorbent assay (ELISA) showed that R-IgG had immunologic reactivity to GII as well as to the GI rV capsid proteins TRX-rV-36, TRX-rV-21, TRX-rV-114, and TRX-rV-96-908. Results of Western immunoblot (WB) analysis showed the same broad recognition of R-IgG when using the same samples. The results of the ELISA tests on serum samples obtained from patients involved in confirmed outbreaks of NLV proved that expressed NLV capsid proteins in E. coli can be detected by NLV-infected human serum. In addition, purified NLVs (LD virus types) derived from patients' stool could be detected using anti-NLV R-IgG, whereas normal R-IgG did not react when using WB. Our results strongly suggest that the immunologic detection of NLV antigens using anti-rV R-IgG is possible and seems a significant step toward simplification of an NLV detection test.
authors.affiliation: Division of Food Microbiology, Osaka Prefectural Institute of Public Health, Osaka, Japan. yoda@iph.pref.osaka.jp
authors.forename: T
authors.initials: T
authors.lastname: Yoda
authors.forename: Y
authors.initials: Y
authors.lastname: Terano
authors.forename: A
authors.initials: A
authors.lastname: Shimada
authors.forename: Y
authors.initials: Y
authors.lastname: Suzuki
authors.forename: K
authors.initials: K
authors.lastname: Yamazaki
authors.forename: N
authors.initials: N
authors.lastname: Sakon
authors.forename: I
authors.initials: I
authors.lastname: Oishi
authors.forename: E T
authors.initials: ET
authors.lastname: Utagawa
authors.forename: Y
authors.initials: Y
authors.lastname: Okuno
authors.forename: T
authors.initials: T
authors.lastname: Shibata
mesh_terms: D018528:ATP-Binding Cassette Transporters; D000818:Animals; D000914:Antibodies, Viral / Q000276:immunology; D001483:Base Sequence; D015153:Blotting, Western / Q000379:methods; D017250:Caliciviridae Infections / Q000097:blood / Q000453:epidemiology / Q000276:immunology* / Q000821:virology; D002213:Capsid / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D036022:Capsid Proteins; D002352:Carrier Proteins / Q000235:genetics / Q000276:immunology; D003001:Cloning, Molecular; D004279:DNA, Viral; D004196:Disease Outbreaks; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D005243:Feces / Q000821:virology; D005759:Gastroenteritis / Q000097:blood / Q000453:epidemiology / Q000276:immunology* / Q000821:virology; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology; D007136:Immunoglobulins / Q000097:blood / Q000276:immunology; D007564:Japan / Q000453:epidemiology; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D009663:Norwalk virus / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D010446:Peptide Fragments / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D013879:Thioredoxins / Q000235:genetics / Q000276:immunology
chemical_list: D018528:ATP-Binding Cassette Transporters; D000914:Antibodies, Viral; D036022:Capsid Proteins; D002352:Carrier Proteins; D004279:DNA, Viral; D029968:Escherichia coli Proteins; D007074:Immunoglobulin G; D007136:Immunoglobulins; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; C121877:Norwalk virus capsid; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C461599:maltose transport system, E coli; D013879:Thioredoxins