Restriction fragment length polymorphism of adhesin genehpaA from differentHelicobacter pyloristrains of Chongqing, China

Abstract

To assess the variability of adhesin gene hpaA between different Helicobacter pylori (H pylori) strains with PCR-restriction fragment length polymorphism (RFLP). Twelve different H pylori strains were chosen to amplify the 710-bp segments of gene hpaA. These strains were NCTC11637, SS1; Chongqing clinical isolates CCS9801, CCS9802, CCS9803, CCS9806, CCS9809, CCS9810, CCS9813, which were gained from patients of gastritis; Mongolia gerbil adapted H pylori strains (abbreviation MG), which were gained from the following steps: gastric mucosal specimens of Mongolia gerbils infected by clinical isolate CCS9803 were cultured and detected, the positive H pylori strains were named as the first generation of Mongolia gerbil adapted H pylori strains (abbreviation MG1) and then were subcultured with healthy Mongolia gerbil to generate MG2, in turn to gain the ninth generation (abbreviation MG9). All hpaA segments, obtained from 12 different H pylori strains, were digested by HhaI and HaeIII individually and analyzed by agarose gel electrophoresis. In all 12 strains, the 710-bp PCR products were successfully amplified and products were cloned to pMD18-T vector respectively, then the recombinant plasmids were digested simultaneously with NcoI and XhoI to recover the small fragments. The objective fragments from 12 different H pylori strains digested with Hae III could be seen as 4 types of bands and 5 types with Hha I. According to the hpaA RFLP patterns, the 12 H pylori strains could be divided into 5 groups: group I, NCTC11637 and SS1; group II, CCS9809, which RFLP type digested with HaeIII was the same as strains of group I, but HhaI RFLP showed difference compared with the other groups; group III, CCS9810; group IV, CCS9803; group V: CCS9801, CCS9802, CCS9806, CCS9813, MG1, MG3 and MG9. The sequence data of 12 hpaA segments were analyzed by DNAsis software and it was observed that: (1) The homologies of base pair and amino acid sequence between strains NCTC11637, SS1, CCS9809 were 99.6% and 98.9%, respectively; (2) The homology of base pair and amino acid sequence between CCS9803 and CCS9810 was 97.7% and 99.1%; (3) That of the rest strains, CCS9801, CCS9802, CCS9806, CCS9813, MG1, MG3, MG9 reached 99.4% and 98.4%; (4) The base pair homologies between all hpaA fragments of different sources were higher than 94.6%, therefore the correspondence of deduced amino acid sequence was higher than 96.8% between each other. The gene hpaA from different H pylori strains revealed variation, and this might provide an effective method for molecular epidemiological survey of H pylori.