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United States Department of Agriculture

Agricultural Research Service

Title: Molecular Cloning, Expression and DNA Sequence Analysis of a Gene Encoding An Outer Membrane Protein of Brachyspria (Serpulina) Pilosicoli

Authors
item Alt, David
item Trott, Darren - AUSTRALIA
item Stanton, Thaddeus

Submitted to: Pig Veterinary Society International Congress Proceedings
Publication Type: Proceedings
Publication Acceptance Date: September 20, 2000
Publication Date: N/A

Technical Abstract: Brachyspira pilosicoli, the causative agent of porcine intestinal spirochaetosis, interacts with the host through end-on attachment to colonic epithelium. Outer membrane proteins may be important to these interactions. Monoclonal antibodies (MAbs) have recently been raised to low density membrane vesicle fractions from B. pilosicoli strain 95-1000. Among 14 strains examined by immunoblot, MAb 2E10 reacted only with 95-1000, identifying a 23 kDa protein which was localized to the outer membrane by immunogold labeling. Surface location of this protein may make it a candidate for development of diagnostic assays. The major objective of this study was to clone, sequence and express the B. pilosicoli protein reactive with MAb 2E10. Two positive clones, p151 and p421 were identified by screening of a genomic library of 95-1000. A 19kDa protein is predicted by a putative 540 bp open reading frame within the sequence of the clones. The protein shows homology to the N-terminus (including the acylation site)of a 16 kDa lipoprotein, smpA, from B. hyodysenteriae. The remainder of the protein sequence was unique and did not show any homology with other proteins in Genbank. Immunoblot with MAb 2E10 to whole cell preparations and cell fractions of E. coli containing p151 identified an approximately 27 kDa band, most prominent in outer membrane fractions. N-terminal amino acid sequence analysis of the protein expressed in E. coli matched the first 12 amino acids of the predicted 19 kDa protein. Additional strains are being examined for presence of the 19 kDa gene. Expression of the 19kDa lipoprotein in E. coli will simplify purification and testing of its immune reactivity with convalescent sera and facilitate possible development of diagnostic assays.

Last Modified: 4/20/2014
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