Cloning and expression of a high-molecular-mass major antigen of Helicobacter pylori: Evidence of linkage to cytotoxin production

M. K.R. Tummuru, T. L. Cover, M. J. Blaser

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581 Scopus citations

Abstract

A high-molecular-mass (120- to 128-kDa) Helicobacter pylori antigen has been associated with peptic ulcer disease. We created a bank of 40,000 random chromosomal fragments of H. pylori 84-183 by using λZapII. Screening of this bank in Escherichia coli XL1-Blue with absorbed serum from an H. pylori- infected person permitted the isolation and purification of a clone with a 3.5-kb insert. Subcloning of this insert (pMC3) permitted the expression of a recombinant H. pylori protein that had a mass of approximately 96 kDa and that was recognized by the human serum. Sera that were obtained from H. pylori-infected persons and that recognized the native 120- to 128-kDa H. pylori antigen recognized the recombinant 96-kDa pMC3 protein to a significantly greater extent than did sera that did not recognize the native H. pylori antigen. All 19 H. pylori isolates producing the 120- to 128-kDa antigen hybridized with pMC3; none of 13 nonproducers did so (P < 0.001). Because all 15 isolates producing the vacuolating cytotoxin hybridized with pMC3, we called the gene cagA (cytotoxin-associated gene). Sequence analysis of pMC3 identified an open reading frame of 859 amino acids, without a termination codon. Parallel screening of a λgt11 library with human serum revealed positive plaques with identical 0.6-kb inserts and sequences matching the sequence of the downstream region of pMC3. To clone the full- length gene, we used the 0.6-kb fragment as a probe and isolated a clone with a 2.7-kb insert from the λZapII genomic library. Nucleotide sequencing of this insert (pYB2) revealed a 785-bp sequence that overlapped the downstream region of pMC3. Translation of the complete nucleotide sequence of cagA revealed an open reading frame of 1,181 amino acids yielding a protein of 131,517 daltons. There was no significant homology with any previously reported protein sequence. These findings indicate the cloning and characterization of a high-molecular-mass H. pylori antigen potentially associated with virulence and with cytotoxin production.

Original languageEnglish (US)
Pages (from-to)1799-1809
Number of pages11
JournalInfection and immunity
Volume61
Issue number5
StatePublished - 1993
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Parasitology
  • Microbiology
  • Immunology
  • Infectious Diseases

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