Identification of the gene encoding BmpB, a 30 kDa outer envelope lipoprotein of Brachyspira (Serpulina) hyodysenteriae, and immunogenicity of recombinant BmpB in mice and pigs.
A gene encoding a 30kDa outer envelope protein of the intestinal spirochaete Brachyspira (Serpulina) hyodysenteriae, was cloned and expressed in Escherichia coli strain XLOLR. Five phagemids containing DNA inserts encoding the protein were established and one clone (pSHA) was sequenced. An 816bp hypothetical open reading frame (ORF) was identified, with a potential ribosome binding site (AGGAG), and putative -10 (TATAAT) and -35 (TTGAAA) promoter regions upstream from the ATG start of the ORF. A 12bp inverted repeat sequence, possibly serving as a transcription terminator, was identified downstream from the TAA stop codon. Analysis of the amino acid sequence identified a 19 residue hydrophobic signal peptide, incorporating a potential signal peptidase cleavage site and membrane lipoprotein lipid attachment site. Further analysis of the amino acid usage of this lipoprotein, designated BmpB, showed its possible outer membrane localisation. Comparison of the gene encoding the lipoprotein, bmpB, with GenBank nucleotide sequences showed that it has homology with the gene (plp3) encoding Plp3, an outer membrane lipoprotein of Pasteurella haemolytica (54% identity in 735bp). Comparison of the deduced amino acid sequence with the SWISS-PROT amino acid database revealed greatest homology with the outer membrane lipoproteins (Plp1, 2, 3) of P. haemolytica (34% identity in 242 aa, 37% identity in 250 aa, and 39% identity in 272 aa, respectively), and lipoproteins (rcsF and lipoprotein-28) of E. coli (40% identity in 267 aa and 36% identity in 263 aa, respectively). Three of the recombinant E. coli clones (pSHA, pSHD, and pSHE) were formalinised and used to immunise mice. A bacterin preparation of one recombinant E. coli clone (pSHA) was used to immunise pigs. Sera from these mice and pigs recognised the 30kDa lipoprotein in outer membrane preparations of B. hyodysenteriae, indicating the immunogenicity of recombinant BmpB. Sera from pigs naturally infected with B. hyodysenteriae also reacted with recombinant BmpB expressed in E. coli.
Lee
B J
BJ
Division of Veterinary and Biomedical Sciences, Murdoch University, Murdoch, Perth, WA 6150, Australia.
La
T
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Mikosza
A S
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Hampson
D J
DJ
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001425:Bacterial Outer Membrane Proteins; D015153:Blotting, Western / Q000662:veterinary; D017401:Brachyspira hyodysenteriae / Q000235:genetics* / Q000276:immunology*; D005243:Feces / Q000382:microbiology; D019084:Fluorescent Antibody Technique, Indirect / Q000662:veterinary; D015964:Gene Expression Regulation, Bacterial; D007114:Immunization / Q000662:veterinary; D008074:Lipoproteins / Q000235:genetics* / Q000276:immunology*; D051379:Mice; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D011994:Recombinant Proteins / Q000276:immunology; D013145:Spirochaetales Infections; D013552:Swine
D000911:Antibodies, Monoclonal; D001425:Bacterial Outer Membrane Proteins; C415945:BmpB protein, Brachyspira hyodysenteriae; D008074:Lipoproteins; D011994:Recombinant Proteins