pylori genes, including peptidyl-prolyl cis–trans isomerase (PPIase), which has been characterized as a virulent factor of Legionella pneumophila and Trypanosoma cruzi (Fischer et al., 1992; Pereira et al., 2002) and is implicated in the regulation of gastric epithelial cell growth and apoptosis (Basak et al., 2005). A total of 64 H. pylori strains were
cultured from gastric biopsies from adult patients. These included 22 cases from gastric cancer patients and 42 from superficial gastritis patients. All samples were obtained with informed consent under a protocol approved by the hospital ethical committee at the China Medical University. Helicobacter pylori were cultured at 5% O2/10% CO2/85% N2, 37oC on brain heart infusion agar (Difco) supplemented Tanespimycin cell line with 7% sheep blood, 0.4% IsoVitaleX, amphotericin B (8 g mL−1), trimethoprim (5 g mL−1) and vancomycin (6 g mL−1). Helicobacter pylori colonies were identified based on their typical morphology, characteristic appearance on Gram staining, a positive urease test and
gene-specific PCR tests. Bacterial DNA was extracted with phenol–chloroformisoamyl alcohol by standard procedures and precipitated by the addition of 1/10 volume of ammonium acetate and 2.5 volume of cold ethanol. After centrifugation, the DNA pellet was washed with 70% ethanol and dissolved in TE buffer [10 Mm Tris-HCl (pH 8.3), 0.1 mmol L−1 EDTA] as we have described previously (Gong et al., 2005). The differences in gene content between the gastric cancer-associated H. pylori strain and the superficial gastritis-associated strain was determined using p38 MAPK inhibitor the PCR-Select™ DNA Substraction Kit (Clontech). To detect gastric cancer-specific second genes, genomic DNA from gastric cancer strain, L301, was used as the tester DNA and genomic DNA from superficial gastritis strain, B975,
was used as the driver DNA. To detect genes that were less abundant or absent in gastric cancer-associated H. pylori strain, genomic DNA from B975 was used as the tester DNA and genomic DNA from L301 was used as the driver DNA. Two micrograms of either tester or driver DNAs were digested to completion with 60 U of AluI (New England Biolabs) for 16 h in 200 μL reaction volumes. The digested products were extracted with phenol, precipitated with ethanol and resuspended in 10 mM Tris-HCl, pH 7.5, at a final concentration of 200 ng μL−1. Two aliquots of the digested tester DNAs were ligated separately to two different adaptors (Adaptor 1, 5′-CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAGGT-3′ and 3′-GGCCCGTCCA-5′; Adaptor 2R, 5′-CTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAGGT-3′ and 3′-GCCGGCTCCA-5′) using T4 DNA ligase (New England Biolabs). Then, 1 μL of each adaptor-ligated tester DNAs was mixed with 2.0 μL of digested driver DNAs and 1.0 μL of 4 × hybridization buffer. The DNA fragments were denatured at 98 °C for 1.5 min, and allowed to anneal at 63 °C for 1.5 h.
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