Isolation and biochemical and molecular analyses of a species-specific protein antigen from the gastric pathogen Helicobacter pylori.
A protein of Mr 26,000 which was present in large quantities in extracts of cells of Helicobacter pylori was purified to homogeneity by ammonium sulfate precipitation followed by gel filtration and reversed-phase chromatography or anion-exchange chromatography. The protein appeared to be associated with the soluble fraction of the cell, and antibodies raised against the protein were reactive with whole-cell lysates of a variety of H. pylori strains in a simple immunodot blot assay. This reaction was species specific. Protein sequence determination of the amino terminus and internal cyanogen bromide fragments and amino acid composition analysis were performed. An oligonucleotide derived from these data was used to clone a fragment encoding most of the coding sequence. Expression in Escherichia coli was dependent on vector promoters. The DNA sequence of the fragment was determined. DNA probes derived from the cloned fragment hybridized to genomic DNA of all H. pylori strains tested, but not to DNAs of Helicobacter mustelae, Wolinella succinogenes, various Campylobacter species, and a panel of gram-negative enteric bacteria. The apparent uniqueness of this protein may be exploited for the development of species-specific diagnostics for this gastric pathogen.
O'Toole
P W
PW
Department of Medical Microbiology, University of Lund, Sweden.
Logan
S M
SM
Kostrzynska
M
M
Wadström
T
T
Trust
T J
TJ
D000595:Amino Acid Sequence; D000906:Antibodies; D000942:Antigens, Bacterial / Q000235:genetics / Q000302:isolation & purification*; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification*; D001483:Base Sequence; D015139:Blotting, Southern; D015153:Blotting, Western; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D003488:Cyanogen Bromide; D004269:DNA, Bacterial / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005798:Genes, Bacterial; D016480:Helicobacter pylori / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification; D014176:Protein Biosynthesis; D013045:Species Specificity; D013270:Stomach / Q000382:microbiology*
D000906:Antibodies; D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D010446:Peptide Fragments; D003488:Cyanogen Bromide