CLONING AND EXPRESSION OF A HIGH-MOLECULAR-MASS MAJOR ANTIGEN OF HELICOBACTER-PYLORI - EVIDENCE OF LINKAGE TO CYTOTOXIN PRODUCTION

A high-molecular-mass (120- to 128-kDa) Helicobacter pylori antigen ha s been associated with peptic ulcer disease. We created a bank of 40,0 00 random chromosomal fragments of H. pylori 84-183 by using lambdaZap II. Screening of this bank in Escherichia coli XL1-Blue with absorbed serum from an H. pylori-infected person permitted the isolation and pu rification of a clone with a 3.5-kb insert. Subcloning of this insert (pMC3) permitted the expression of a recombinant H. pylori protein tha t had a mass of approximately 96 kDa and that was recognized by the hu man serum. Sera that were obtained from H. pylori-infected persons and that recognized the native 120- to 128-kDa H. pylori antigen recogniz ed the recombinant 96-kDa pMC3 protein to a significantly greater exte nt than did sera that did not recognize the native H. pylori antigen. All 19 H. pylori isolates producing the 120- to 128-kDa antigen hybrid ized with pMC3; none of 13 nonproducers did so (P < 0.001). Because al l 15 isolates producing the vacuolating cytotoxin hybridized with pMC3 , we called the gene cagA (cytotoxin-associated gene). Sequence analys is of pMC3 identified an open reading frame of 859 amino acids, withou t a termination codon. Parallel screening of a lambdagt11 library with human serum revealed positive plaques with identical 0.6-kb inserts a nd sequences matching the sequence of the downstream region of pMC3. T o clone the full-length gene, we used the 0.6-kb fragment as a probe a nd isolated a clone with a 2.7-kb insert from the lambdaZaplI genomic library. Nucleotide sequencing of this insert (pYB2) revealed a 785-bp sequence that overlapped the downstream region of pMC3. Translation o f the complete nucleotide sequence of cagA revealed an open reading fr ame of 1,181 amino acids yielding a protein of 131,517 daltons. There was no significant homology with any previously reported protein seque nce. These findings indicate the cloning and characterization of a hig h-molecular-mass H. pylori antigen potentially associated with virulen ce and with cytotoxin production.