title: Immunological properties of recombinant porin of Haemophilus influenzae type b expressed in Bacillus subtilis.
abstract: The major surface-located, channel-forming protein in the outer membrane of Haemophilus influenzae type b (Hib) is porin (341 amino acids; M(r), 37,782). In order to generate Hib porin that is devoid of lipooligosaccharides and capsular polysaccharide, the Hib porin gene ompP2 was subcloned into a plasmid vector and recombinant Hib porin was expressed in Bacillus subtilis. Recombinant porin was produced in large quantities in B. subtilis and formed intracellular inclusion bodies. Recombinant porin was extracted from inclusion bodies and shown to be active in forming pores in synthetic black lipid membranes. However, these pores demonstrated different pore characteristics than wild-type Hib porin. Mouse hyperimmune sera against recombinant porin were generated and subjected to epitope scanning with a library of 336 overlapping synthetic hexapeptides that corresponded to the entire sequence of Hib porin. The epitope specificities of the anti-recombinant porin antibodies were similar to those of antibodies against Hib porin: selected regions near the amino terminus which include a buried loop in the native structure of Hib porin were more immunogenic than regions at the carboxy terminus. Although some mouse anti-recombinant porin antibodies mediated complement-dependent binding to Hib by polymorphonuclear leucocytes in opsonophagocytosis assays, the antibodies were not bactericidal, nor did they abrogate bacteremia in the infant rat model of infection. It was concluded that the native state of Hib porin is required for the generation of a protective immune response against the bacterium.
authors.affiliation: Department of Microbiology and Immunology, McGill University, Montreal, Quebec, Canada.
authors.forename: R
authors.initials: R
authors.lastname: Srikumar
authors.forename: D
authors.initials: D
authors.lastname: Dahan
authors.forename: M F
authors.initials: MF
authors.lastname: Gras
authors.forename: L
authors.initials: L
authors.lastname: Saarinen
authors.forename: H
authors.initials: H
authors.lastname: Käyhty
authors.forename: M
authors.initials: M
authors.lastname: Sarvas
authors.forename: L
authors.initials: L
authors.lastname: Vogel
authors.forename: J W
authors.initials: JW
authors.lastname: Coulton
mesh_terms: D000818:Animals; D000907:Antibodies, Bacterial / Q000032:analysis / Q000276:immunology; D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism; D001425:Bacterial Outer Membrane Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D001483:Base Sequence; D000939:Epitopes / Q000032:analysis; D006193:Haemophilus influenzae / Q000235:genetics / Q000276:immunology*; D007473:Ion Channels; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D018272:Porins; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology
chemical_list: D000907:Antibodies, Bacterial; D001425:Bacterial Outer Membrane Proteins; D000939:Epitopes; D007473:Ion Channels; D018272:Porins; D011994:Recombinant Proteins