Helicobacter Pylori Screening Of Stomach-related Protein And Its Pathogenic Mechanism Of A Preliminary Study

Helicobacter pylori is a micro-aerophilic,Gram-negative bacterium that efficiently colonizes the human stomach mucosa. H. pylori is estimated to infect at least half of the world's population, and chronic, persistent infection by H. pylori may cause gastric diseases, such as chronic atrophic gastritis, peptic ulcers, mucosa-associated lymphoid tissue (MALT) lymphoma or even adenocarcinoma. Many H. pylori virulence factors have been identified and characterized as either direct or indirect causes of observed pathophysiologies. Most studies have focused on the cagA gene and several reported that cagA positive strains of H. pylori are associated with an increased risk for development of atrophic gastritis and gastric adenocarcinoma. Asian populations have not confirmed this association. There may be other genes that are important in the pathogenesis of gastric disease. The aim of this study were to identity new gastric disease–related antigens from H.pylori and characterize their role in the pathogenesis preliminarily.First, twenty candidate genes were selected from some pioneer studies.H.pylori SS1 genomic DNA was used as template for PCR- amplification of genes. The product were cloned into His-tag express vector, pET22b (Novagen Corp). Sixteen His-tagged recombinant proteins were expressed in Escherichia coli BL21 with 1 mM IPTG for 4 h, and then purified using Ni2+-loaded His-Trap Chelating column. The purity of the recombinant proteins reached or exceeded 80% .Serum of high antibody titer was also raised from mice immunized with these recombinant proteins.In order to identify the 16 proteins whether correlate with clinical outcome , a case-control study comparing their seroprevalence of antibodies with Gastric cancer (GC) and chronic gastritis(CG) population was carried out using enzyme -linked immunosorbent assay (ELISA).The proteins showing higher seroreacticity of recognition in GC group compared with CG are HP0762 (OR=6.61, 95%CI 2.70～16.16), HP1424(OR=3.58, 95%CI 1.23～10.48) and HP0232 (OR=2.23,95%CI 1.14～4.35), The seroprevalence among 91 gastric cancer case were 44.0% ,72.5%,52.7% respectively. HP0110, HP0305, HP0596 were identified to be associated with the presence of chronic gastritis. The positive value and OR were: HP0110: 57.6%,OR=8.14( 95%CI 3.88～17.09); HP0305:31.7, OR=3.75, (5%CI 1.37～10.28), HP0596: 66.7%, OR=10.13, (95%CI 4.41～23.28). This studies suggested that person infected H.pylori with these three genes have higher risks of gastric inflammation.Helicobacter pylori infection generally induces local inflammation in the stomach associated with induction of proinflammatory cytokines, such as tumor necrosis factor-α(TNF-α), interleukine-1 (IL-1), and IL-8. Here, we demonstrated that HP0596 could stimulate macrophage to produce TNF-α. The TNF-α-inducing ability of tHP0596, which lacks two cysteines in the N-terminal domain, was impaired compared with HP0596. tHP0596 lose the capability to form a homodimer, cannot enforced expression of TNF-α. While we found HP0596 induce NF-кB activation. Thus, we considered HP0596 was a H.pylori virulence factor associated with gastritis, which can enforced expression of TNF-αby the NF-кB pathway.