Tummuru M K, Cover T L, Blaser M J
Department of Medicine, Vanderbilt Unviersity School of Medicine, Nashville, Tennessee 37232-2605.
Infect Immun. 1993 May;61(5):1799-809. doi: 10.1128/iai.61.5.1799-1809.1993.
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.
一种高分子质量（120至128 kDa）的幽门螺杆菌抗原与消化性溃疡病有关。我们利用λZapII构建了一个包含40000个幽门螺杆菌84 - 183随机染色体片段的文库。用来自幽门螺杆菌感染者的吸收血清在大肠杆菌XL1 - Blue中筛选该文库，使得一个带有3.5 kb插入片段的克隆得以分离和纯化。该插入片段（pMC3）的亚克隆允许表达一种重组幽门螺杆菌蛋白，其质量约为96 kDa，且能被人血清识别。从幽门螺杆菌感染者获得的、能识别天然120至128 kDa幽门螺杆菌抗原的血清，比不能识别天然幽门螺杆菌抗原的血清能更显著地识别重组96 kDa的pMC3蛋白。所有19株产生120至128 kDa抗原的幽门螺杆菌分离株都与pMC3杂交；13株不产生该抗原的分离株均未杂交（P < 0.001）。由于所有15株产生空泡细胞毒素的分离株都与pMC3杂交，我们将该基因命名为cagA（细胞毒素相关基因）。pMC3的序列分析确定了一个859个氨基酸的开放阅读框，没有终止密码子。用人血清对λgt11文库进行平行筛选，发现了具有相同0.6 kb插入片段且序列与pMC3下游区域序列匹配的阳性噬菌斑。为了克隆全长基因，我们用0.6 kb片段作为探针，从λZapII基因组文库中分离出一个带有2.7 kb插入片段的克隆。该插入片段（pYB 2）的核苷酸测序揭示了一个785 bp的序列，与pMC3的下游区域重叠。cagA完整核苷酸序列的翻译揭示了一个1181个氨基酸的开放阅读框，产生一种131517道尔顿的蛋白质。与任何先前报道的蛋白质序列均无显著同源性。这些发现表明克隆并鉴定了一种可能与毒力和细胞毒素产生相关的高分子质量幽门螺杆菌抗原。
