IDENTIFICATION AND CHARACTERIZATION OF AN IMMUNOGENIC OUTER-MEMBRANE PROTEIN OF CAMPYLOBACTER-JEJUNI

We cloned and expressed in Escherichia coli a gene encoding an 18-kDa outer membrane protein (Omp18) from Campylobacter jejuni ATCC 29428, T he nucleotide sequence of the gene encoding Omp18 was determined, and an open reading frame of 165 amino acids was revealed, The amino acid sequence had the typical features of a leader sequence and a signal pe ptidase ZI cleavage site at the N-terminal part of Omp18, Moreover, th e sequence had a high degree of similarity to the peptidoglycan-associ ated outer membrane lipoprotein PG of Haemophilus influenzae and the p eptidoglycan-associated lipoprotein PAL off. coli, Southern blot analy sis in which the cloned gene was used as a probe revealed genes simila r to that encoding Omp18 in all species of the thermophilic group of c ampylobacters as well as Campylobacter sputorum. All campylobacters te sted expressed a protein with a molecular mass identical to that of Om p18, The protein reacted immunologically with polyclonal antibodies di rected against Omp18 from C. jejuni. PCR amplification of the gene enc oding Omp18 with specific primers and subsequent restriction enzyme an alysis of the amplified DNA fragments showed that the gene for Omp18 i s highly conserved in C, jejuni strains isolated from humans, dogs, ca ts, calves, and chickens but is different in other Campylobacter speci es, In order to obtain pure recombinant Omp18 protein for serological assays, the cloned gene for Omp18 was genetically modified by replacin g the signal sequence with a DNA segment encoding six adjacent histidi ne residues, Expression of this construct in E, coli allo,vcd purifica tion of the modified protein (Omp18-6xHis) by metal chelation chromato graphy. Sera from patients with past C, jejuni infection reacted posit ively with Omp18-6xHis, while sera from healthy blood donors showed no reaction with this antigen, Omp18, which is an outer membrane protein belonging to the Family of PALs is well conserved in C, jejuni and is highly immunogenic, It is therefore a good candidate as an antigen fo r the serological diagnosis of past C,jejuni infections.