Research Of Structure And Function About HP0596 From Helicobacter Pylori

Helicobacter pylori is a micro-aerophilic, Gram-negative bacterium that colonizes the human stomach mucosa, which may cause gastric diseases, such as chronic atrophic gastritis, peptic ulcers, mucosa-associated lymphoid tissue (MALT) lymphoma or even adenocarcinoma. And it was classified as Type I carcinogen by the World Health Organization. There are some drawbacks with the current antibiotic therapies against H.pylori, so it was very important and necessary to develop an effective and protective H.pylori vaccine. The widely used antigens include H.pylori whole cell lysate, urease, catalase et al were not good, consequently, the search of some new protective antigens is needed.The HP0596 maybe a possible membrane protein which has a lipoprotein box by the analysis of bioinformatic. It also shows a correlation with gastritis and gastric cancer, so HP0596 maybe a pathogenic factor from H.pylori which is related to stomach diseases. The aims of this study were to identity the position of this protein and explain its function in the pathogenesis preliminarily.First, H.pylori 26695 genomic DNA was used for the template for PCR-amplification of genes hp0596 and the signal peptide, then linked the gene of the signal peptide and the EGFP as a unity. And each was constructed into prokaryotic expression vector plasmid pET-22b from Novagen Corp. Recombinant proteins were expressed in E.coli BL21 (DE3). Then, fluorescence of the EGFP was displaied in the membrane of the E.coli. Periplasmic protein and supernatant protein were used for western blot to confirm the position.HP0596N and HP0596NT whose Cysteine mutate into Serine, neither of the protein has the signal peptide. And each was constructed into prokaryotic expression vector plasmid pET-22b(+) from Novagen Corp. His-tagged recombinant proteins were expressed in E.coli BL21, and then purified using Ni2+-loaded His-Trap Chelating column. The recombinant proteins was puritied which reached or exceeded 90%. The polyclonal antibody against this recombinant protein HP0596N with high titer was obtained.In the end, the recombinant proteins stimulated mouse macrophage cell RAW264.7 that produced TNF-a, IL-6, IFN-y and IL-1β.HP0596NT did not lose the activity of pro-inflammatory factors, and the dimer will enhance the biological activity. Results of the above will lay the foundation for further research of the HP0596 function.