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

@article{citeulike:2626656, 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 lambda ZapII. 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 lambda gt11 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 lambda ZapII genomic library. Nucleotide sequencing of this insert (pYB 2) 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.}, address = {Department of Medicine, Vanderbilt Unviersity School of Medicine, Nashville, Tennessee 37232-2605.}, author = {Tummuru, M. K. and Cover, T. L. and Blaser, M. J. }, citeulike-article-id = {2626656}, issn = {0019-9567}, journal = {Infection and immunity}, keywords = {historical, pylori}, month = {May}, number = {5}, pages = {1799--1809}, posted-at = {2008-04-03 17:10:42}, priority = {2}, title = {Cloning and expression of a high-molecular-mass major antigen of Helicobacter pylori: evidence of linkage to cytotoxin production.}, url = {http://view.ncbi.nlm.nih.gov/pubmed/8478069}, volume = {61}, year = {1993} }

TY - JOUR ID - citeulike:2626656 L3 - citeulike-article-id:2626656 TI - Cloning and expression of a high-molecular-mass major antigen of Helicobacter pylori: evidence of linkage to cytotoxin production. JF - Infection and immunity VL - 61 IS - 5 SP - 1799 EP - 1809 AD - Department of Medicine, Vanderbilt Unviersity School of Medicine, Nashville, Tennessee 37232-2605. SN - 0019-9567 N2 - 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 lambda ZapII. 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 lambda gt11 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 lambda ZapII genomic library. Nucleotide sequencing of this insert (pYB 2) 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. KW - historical KW - pylori AU - Tummuru, MK AU - Cover, TL AU - Blaser, MJ PY - 1993/05// UR - http://view.ncbi.nlm.nih.gov/pubmed/8478069 ER -