| Objective To analyze comparativelythe structural characteristics of cytotoxinassociated gene A protein(Cag A)from East Asia strains and Western strains of Helicobacter pylori(H.pylori), respectively, by isolation and identification of the clinical strains of H.pylori and analysis of the phosphorylation motif such as EPIYA in Cag A protein, and thento investigate preliminarily the effect of the East Asia- and Western- type Cag A on proliferation and apoptosis of gastric epithelial cell. Methods Gastroscopic mucosa biopsies from the patients with gastric diseases were acquired and cut into pieces, and then these pieces were inoculated to Columbia blood agar plate with selective medium and cultured under the microaerobic condition. The bacterial colony was preliminarily identified using urease test, gram stain and microscopic examination. DNA of the bacteria was abstracted and used to amplify H. pylori 16 S r RNA geneby polymerase chain reaction(PCR), and the products were identified by sequencing.The cag A gene of H. pylori was amplified and connected into p MD-18 T vector. The recombinant vectors were transformed into competent E. coli DH5 and the positive clones were selected, and the plasmid DNA of these positive clones was abstracted and identified by sequencing. Finally, the DNAStar V7.1 and MEGA 6 softwares were used to analyze Cag A sequences to construct the molecular phylogenic trees of Cag A. H.pylori Cag A proteins were analyzes by bioinformatics using the open bioinformation databases on line and a variaty o fbiology softwares performing to predict the structure and functions.Thecag A gene of East Asian and Western strains were respectively cloned into the eukaryotic expression vector pc DNA3.1 and identified by sequencing. Gastric cancer cell line MKN-45 cells were transfected with pc DNA3.1/cag A and pc DNA3.1 vectors by Lipofectamine2000 TMrespectively, and then the cell proliferation and apoptosis were detected by flow cytometyr. Results A total of 26 clinical strains of H. pylori were successfully isolated and the recombinant p MD18-T/cag A vectors were successfully constructed and sequenced. All sequences were submitted to Gen Bankand their accession numbers are KR154731~KR154758. By sequence alignment using blast software and clustering analysis, we found that 6 of 26 H. pylori strains were Western type and the remaining 20 strains were East Asia type.The deletion, partial deletion and variation of 13 amino acids at the 834-815 th amino acid sites of Cag A from East Asia strains were detected and the deletions of EPIYA-C motif of Cag A from Western strains were discovered. Clustering analysis showed that the clinical H.pylori strains were clustered into East Asian group, East Asia of Western-type group and Western group. Further, it was found that the isolates have the highest homologywith the Japanese strains, and the strains are clustered together into East Asia group. Cag A/26695(Western strain) protein contains 1184 amino acids and its molecular weight is about 132.36 k D, as well as the theory isoelectric point is 8.93. Cag A/GZ7(East Asia strain) protein contains 1175 amino acids and its molecular weight is about 131.32 k D, as well as the theory isoelectric point is 8.30. Bioinfomatics analysis showed that the spatial structure of Cag A from East Asia and Western strains were similar, and both were hydrophilic proteins in the cytoplasm. Howevertheposition of antigenic peptides, the composition of the secondary structure and the conserved domains in the Cag A protein from are different between the East Asian and the western strains.Compared with control pc DAN3.1 group, the apoptosis rateof the pcag A / 26695 and pcag A / GZ7 groups with high cag A-expression is(8.29 ± 0.61) % and(9.98 ± 1.07) % repectively, which are significantly higher than pc DNA3.1 group(5.38 ± 0.61) %(all P<0.05). There was not statistically significant between pcag A /26695 group and pcag A /GZ7 group(P>0.05). The percentage of cells in G0-G1 phase in pcag A/26695 and pcag A/GZ7 groups were significantly higher than pc DNA3.1 group(36.14 ± 3.16,36.56 ± 1.62 and 30.88 ± 1.03, respectively), and the percentage of S phase cells weresignificantly lower than pc DNA3.1 group(48.57 ± 2.90,48.01 ± 2.22 and 54.57 ± 1.52, respectively)(all P<0.05).The G2-M phase cells have no difference among three groups(15.29 ± 0.43,15.42 ± 0.59 and 14.55 ± 0.88, respectively)(P>0.05). Conclusion The East Asia H. pylori are a majority in the isolated clinical strains, but there is also Western strain. There are the structural differences between the East Asia and Western strains. Bioinformatics prediction showed that there are some differences in the secondary structure and conserved domain of the two-type Cag As, but there is no significant difference in the tertiary structure.Cag A can inhibit the cell proliferation and promote the cell apoptosis of gastric cancer cells, but the role of East-Asia- and Western-type Cag A has not significant difference. |
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