Cloning and expression of a Helicobacter bilis immunoreactive protein

Abstract

In an effort to identify immunoreactive Helicobacter bilis antigens with potential for serodiagnosis, sera from mice experimentally infected with H. bilis were used to screen an H. bilis genomic DNA expression library. Among 17 immunoreactive clones, several contained sequences that encoded a predicted 167-kDa protein (P167). Five overlapping P167 peptides (P167A to P167E) of approximately 40 kDa each were generated and tested. Immune sera reacted with fragments P167C and P167D at dilutions of 1:1,600 and 1:6,400, respectively, and reacted with an H. bilis membrane extract at a dilution of 1:800 in an enzyme-linked immunosorbent assay. Sera from mice experimentally infected with H. hepaticus did not react with P167C and P167D. Sera from mice naturally infected with H. bilis but not sera from mice naturally infected with H. hepaticus reacted with P167C and P167D. Hyperimmune sera against P167C peptide reacted with recombinant P167C and with a 120-kDa band in H. bilis lysates but did not react with a protein of the same size on immunoblots prepared from H. hepaticus, H. muridarum, or unrelated Borrelia burgdorferi and Campylobacter jejuni whole-cell lysates. Nevertheless, the P167A, P167B, P167C, and P167D primers, but not the P167E primers, amplified DNA from H. hepaticus, and all five primer sets amplified DNA from H. muridarum. These results suggest that P167 is an immunodominant, H. bilis-specific antigen that may have potential for use in serodiagnosis.

FIG. 1.

Immunoblots of an H. bilis membrane extract and recombinant proteins probed with H. bilis or H. hepaticus immune sera. Lane 1, H. bilis membrane extract probed with H. bilis immune serum. Lane 2, P167C probed with H. bilis immune serum. The asterisk indicates P167C, and the lower bands are probably degraded products of P167C. Lane 3, P167C probed with H. hepaticus immune serum. Lane 4, P167D probed with H. bilis immune serum. The asterisk indicates P167D, and the lower bands are probably degraded products of P167D. Lane 5, P167D probed with H. hepaticus immune serum.

FIG. 2.

Relative sizes, interrelationships, and locations of peptides P167A to P167E in relation to the entire P167 molecule.

FIG. 3.

PCR amplification of the P167 fragments with H. bilis (lanes 1 to 5), H. hepaticus (lanes 6 to 10), and H. muridarum (lanes 11 to 15) targets. Lanes 1, 6, and 11, P167A DNA. Lanes 2, 7, and 12, P167B DNA. Lanes 3, 8, and 13, P167C DNA. Lanes 4, 9, and 14, P167D DNA. Lanes 5, 10, and 15, P167E DNA.

FIG. 4.

Dot blot with sera from mice naturally infected with H. bilis or H. hepaticus. The top row contains H. bilis membrane antigen extract; the middle row contains recombinant P167C antigen; and the bottom row contains recombinant P167D antigen. Lanes 1 to 9 contain sera from nine mice naturally infected with H. bilis; lanes 10 to 13 contain sera from four mice naturally infected with H. hepaticus.