title: Identification of an immunogenic 18-kDa protein of Helicobacter pylori by alkaline phosphatase gene fusions.
abstract: The use of alkaline phosphatase fusion methodology to identify Helicobacter pylori exported proteins enabled the identification of an open reading frame (ORF) encoding a highly immunogenic, previously uncharacterised exported protein. The predicted aminoacid sequence displays a typical N-terminal signal peptide and contains regions of C-terminal hydrophobicity consistent with a membrane-associated protein. Southern blot analysis revealed that the gene encoding the protein was absent in several Helicobacter spp. and a combination of PCR and sequence analysis of the amplified gene showed that it is highly conserved amongst isolates of H. pylori. To obtain pure recombinant protein, the gene encoding the protein was cloned and expressed as a beta-galactosidase (beta-gal) fusion in Escherichia coli and the protein was purified by affinity chromatography and proteolytic cleavage of the beta-gal portion. The purified protein, which has an apparent mol. wt of 18 kDa, was recognised by antibody present in 71% of sera from patients infected with H. pylori, but in only 16% of sera from patients with unrelated or no gastrointestinal disease, by Western blot assays. These results indicate that the 18-kDa protein from H. pylori is immunogenic and is expressed in vivo.
authors.forename: Jane
authors.initials: J
authors.lastname: Oliaro
authors.forename: Richard D
authors.initials: RD
authors.lastname: Johnson
authors.affiliation: Institute of Veterinary, Animal and Biomedical Sciences, Massey University, Palmerston North and *Wakefield Gastroenterology Research Centre, Wakefield Hospital, Wellington, New Zealand.
authors.forename: Wangxue
authors.initials: W
authors.lastname: Chen
authors.affiliation: Institute of Veterinary, Animal and Biomedical Sciences, Massey University, Palmerston North and *Wakefield Gastroenterology Research Centre, Wakefield Hospital, Wellington, New Zealand.
authors.forename: Vinton S
authors.initials: VS
authors.lastname: Chadwick
authors.forename: Alan
authors.initials: A
authors.lastname: Murray
mesh_terms: D000469:Alkaline Phosphatase / Q000235:genetics; D000595:Amino Acid Sequence; D000942:Antigens, Bacterial / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D001426:Bacterial Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D003001:Cloning, Molecular / Q000379:methods; D016480:Helicobacter pylori / Q000276:immunology*; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D011993:Recombinant Fusion Proteins / Q000276:immunology / Q000302:isolation & purification; D013045:Species Specificity
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D000469:Alkaline Phosphatase