| Vibrio cholerae is a gram-negative enteric bacterium that causes cholera epidemics worldwide. The only vaccine approved for mass vaccination at the present time provides short term protection, but people who recover from cholera have long-term immunity. Most of the prolonged immunity is directed against the outer membrane proteins (OMPs) of V. cholerae . After studying the OMP profile of V. cholerae V86 El Tor Inaba, four OMPs (43, 42, 30 and 22 kDa) were identified and their amino termini were sequenced. The gene encoding the 22 kDa OMP, ompw , had been previously cloned. Using specific primers, the ompw gene was PCR-amplified and cloned into the pT7-6 expression vector. The recombinant protein was visualized by Western blot analysis and by 35S-methionine labeling. Total cellular protein was analyzed by electrophoresis using a PrepCell and the fractions containing OMPW were identified by Western blot and pooled. The flagellin core protein of V. cholerae was isolated and the N-terminal sequence was determined. The amino acid sequence exhibited significant homology with the Pseudomonas aeruginosa flagellin core protein (FlaA). A synthetic oligonucleotide was designed using the amino acid sequence of the V. cholerae flagellin and the known nucleotide sequence of the P. aeruginosa flaA gene. This oligonucleotide was used to screen a V. cholerae genomic DNA library constructed in λgt11. Recombinant phage particles that hybridized to the probe were identified and the DNA was isolated from them. These recombinants contained a 5.6 kb V. cholerae genomic DNA fragment which was amplified by PCR using primers to the λgt11 vector and cloned into a plasmid vector, pBluescript. When the DNA fragment was sequenced, it contained the partial flaA gene (596 bp) at the 3′ end and three other open reading frames (ORFs) upstream. The ORFs were homologous to flagellar genes cloned from other Vibrio species and were identified and named as flgL, M and J that encoded the hook-associated proteins (HAPs) 3 and 1 and the L-ring protein, respectively, of the flagellum. In addition, the 5 ′ end of the 5.6 kb fragment contained a partial ORF (364 bp) that was identified as flgl and encoded the P-ring protein. The complete flaA gene was cloned from V. cholerae genomic DNA, using the 596 bp fragment as a probe and sequenced. The flaA gene was 1.2 kb and encoded a protein of 42 kDa. The complete flgI gene was cloned using the 364 bp partial gene fragment as a probe. The flgL, M, J and I genes encoded proteins of 44, 69, 37, and 38 kDa, respectively. (Abstract shortened by UMI.)... |
