Characterization Of 0521 Gene In The Cag Pathogenicity Island Of Helicobacter Pylori

hp0521 is the second gene of cag PAI in the type I Helicobacter pylori, and at present, the research of this gene remains on the sequence diversity and its influence on the Cag A transport, as well as the IL-8 secretion across the world. This experiment aims at the diversity and function of hp0521 gene in the H.pylori, and does some research on its role in the pathogenesis of H.pylori from bioinformatics, molecular biology and so on.Methods:1. We clone and sequence the hp0521 gene and cag A gene 3,variable region through the PCR amplification in the NCTC11637 strain and clinical isolates of H.pylori. Analyze the basic physicochemical properties and construct the phylogenetic tree of hp0521 gene by using bioinformatics software. At the same time, we explore the relationship between hp0521 genotype and cag A 3,variable region sequence.2. Based on the H.pylori strain OM6004 hp0521 gene, we acquire the strain 26695 new CDS(coding sequence)with completion two bases by vitro synthesis, then construct expression vector p ET-32a-hpc0521 and p ET-32a-hp0521. Finally, we bestow IPTG to induce the expression of the fusion protein and Western Blot to identify.3. We analyze hp0521 gene sequence antigenic determinant of H.pylori strain 26695 and NCTC11637, meanwhile, adopt the synthesis of peptides with strong antigenic to immune and obtain the antibody, measuring titer as well.4. Verify the expression of hp0521 gene encoding Cag2 protein in the H.pylori strain26695 by Western Blot.5. Construct the suicide plasmid p Blue KM40-△hp0521:: Kan and apply electric transfer, resistance screening, PCR, sequencing, to obtain the defective strain △hp0521.6. Measure OD(optical density)values depending on the time point and compare the wild strain 26695 growth to the defective strain △hp0521.7. Extract the total RNA of the wild strain 26695 and defective strain △hp0521with reverse transcription, and compare the polarization of downstream gene hp0522 and hp0523 by PCR.8. Search for the hp0521 interaction genes by website prediction and literature, and design RT-PCR primers to compare the changes on the level of m RNA.9. Confirm the stability of other important cag PAI encoding protein by Western Blot in the wild strain 26695 and defective strain △hp0521.10. Make HP co-culture with GES-1 and compare the changes of Cag A transport as well as IL-8 secretion with the deletion of hp0521 gene.Results:1. We gain the hp0521 gene and cag A gene 3,variable region sequence of H.pylori strain. And the hp0521 gene encoding stable Cag2 protein without signal peptide, as well as the main structural component consists of random coil and alpha helix. Cag2 protein has high homology with the DNA topoisomerase I, with the mediated cell wall extension tag protein and so forth. We obtain its homologous strains of hp0521 gene by the phylogenetic analysis.2. We synthetize successfully completion base CDS of H.pylori strain 26695, and construct the prokaryotic expression vector p ET-32a-hpc0521, as well as the p ET-32a-hp0521. It can be determined that hpc0521 gene is able to encode Cag2 protein by Western Blot.3. Rabbit anti-Cag2 protein polyclonal antibody is obtained, and the titer was1:2.56×105.4. Using rabbit anti-Cag2 protein polyclonal antibody, Western Blot determines that H.pylori strain 26695 hp0521 gene does not encode Cag2 protein.5. Construct suicide plasmid p Blue KM40-(35)hp0521::kan successfully, and with the successful transformation, we obtain the defective strain △hp0521.6. Drawing growth curve of the wild strain 26695 and defective strain △hp0521,determines that the hp0521 gene does not affect its growth.7. Determine that the deletion of hp0521 gene has no effect on the hp0522 and hp0523 gene transcription.8.Acquire the hp0521 interaction gene, such as hp0116、hp0333、hp0440、hp0515、hp0516、hp0520、hp0792、hp1293、hp1324、rpo B、cag A、vac A、ure A、fla B, and determine that the deletion of hp0521 gene can level up cag A m RNA expression by RT-PCR, as well as rpo B.9. With the hp0521 gene deletion, expression of the cag PAI encoded Cag A protein increases, and does not affect the protein stability of Cag X、Cag M、Cag L、Cag S、Cag I、Cag V、Cagζ.10. As the co-culture with GES-1, there is no significant change in Cag A translocation and IL-8 induction with the deletion of hp0521 gene.Conclusion:1. We understand the hp0521 gene encoding Cag2 protein with DNA topoisomerase function area by cloning, sequencing and analysis. It is concluded that cag A gene of H.pylori carried by the EPIYA-D motif generally deletes hp0521 gene.2. We validate successfully that the hp0521 gene does not encode Cag2 protein and have found the reason that the deletion of two bases causes the termination codon in advance.3. The hp0521 gene deletion having no effect on the growth of H.pylori and the stability of other cag PAI encoding protein is determined, as well as Cag A transport and IL-8 induced secretion.6. It is proved that the hp0521 gene may interact with cag A and rpo B, at the same time, the hp0521 gene may inhibit the production of Cag A protein.