Mkr. Tummuru et al., CLONING AND EXPRESSION OF A HIGH-MOLECULAR-MASS MAJOR ANTIGEN OF HELICOBACTER-PYLORI - EVIDENCE OF LINKAGE TO CYTOTOXIN PRODUCTION, Infection and immunity, 61(5), 1993, pp. 1799-1809
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.