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 30 kDa outer envelope protein of the intestinal spirochae te Brachyspira (Serpulina) hyodysenteriae, was cloned and expressed in Esch erichia coli strain XLOLR. Five phagemids containing DNA inserts encoding t he protein were established and one clone (pSHA) was sequenced. An 816 bp h ypothetical open reading frame (ORF) was identified, with a potential ribos ome binding site (AGGAG), and putative -10 (TATAAT) and -35 (TTGAAA) promot er regions upstream from the ATG start of the ORE A 12 bp inverted repeat s equence, possibly serving as a transcription terminator, was identified dow nstream from the TAA stop codon. Analysis of the amino acid sequence identi fied a 19 residue hydrophobic signal peptide, incorporating a potential sig nal peptidase cleavage site and membrane lipoprotein lipid attachment site. Further analysis of the amino acid usage of this lipoprotein, designated B mpB, showed its possible outer membrane localisation. Comparison of the gen e 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 735 bp). 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 3 9% identity in 272 aa, respectively), and lipoproteins (rcsF and lipoprotei n-28) of E. coli (40% identity in 267 aa and 36% identity in 263 aa, respec tively). Three of the recombinant E. coli clones (pSHA, pSHD, and pSHE) wer e formalinised and used to immunise mice. A bacterin preparation of one rec ombinant E. coli clone (pSHA) was used to immunise pigs. Sera from these mi ce and pigs recognised the 30 kDa lipoprotein in outer membrane preparation s 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. (C) 2000 Elsevier Science B.V. All r ights reserved.