Prokaryotic Expression Of Helicobacter Pylori CheA And CheY Genes And Correlation Among The Expressed Products And Bacterial Chemotactic Behavior

Background and Objective Helicobacter pylori infection is closelyrelated to the incidence of diseases such as human gastritis, peptic ulcer, gastric adenocarcinoma.The pathogenesis of most bacteria is chemotaxis and colonization in the appropriate parts of the host parasite,and then causes disease by a large number of breeding.In recent years, it was found that bacterial chemotaxis is controlled by the two-component signal systems (TCS).CheA / CheY is H. pylori chemotaxis-related TCS, mutant lack of CheA and CheY despite have power, but not colonization in mouse gastric mucosa,suggesting that Che-TCS in bacteria play a key role in colonization.However,the activation of Helicobacter pylori Che-TCS signal and its blocking drugs have not been reported,the Che-TCS working mechanisms are still to be in-depth study.In this study, we cloned the H. pylori NCTC11637 strain of CheA and CheY genes and construct a prokaryotic expression system,prepared the purpose of recombinant expression products rCheA and rCheY antisera and IgG, using a hard agar plus (HAP) to establish chemotaxis model in vitro of Helicobacter pylori,and chemokine inducers were screened of Helicobacter pylori in vitro with a view to further clarify the Helicobacter pylori Che-TCS signal transduction and its regulation mechanism.Methods The segments of entire cheA and cheY genes were amplified by PCRand then sequenced after T-A cloning. Prokaryotic expression systems of the genes were subsequently constructed. SDS-PAGE plus BioRad Gel Image Analyzer were used to examine the expression of target recombinant proteins rCheA and rCheY, and Ni-NTA affinity chromatography was performed to extract rCheA and rCheY. Rabbits were immunized with rCheA and rCheY to obtain antisera and IgG in each of the antisera was extracted by saturated ammonium sulfate precipitation and DEAE-32 ion exchange chromatography. Immunodiffusion assay was performed to measure the titers of antisera and their IgGs. Chemotactic model in vitro of H. pylori based on hard-agar plus method was established to determine the chemotaxis-inducing effects of eleven candidate substances. Simultaneously, the effects of rCheA-IgG and closantel sodium on blocking the bacterial chemotactic behavior were also observed.Results The segments with expected sizes of cheA and cheY genes wereobtained by PCRs, and their nucleotide and putative amino acid sequences were 100% idenities to the reports. The constructed prokaryotic systems could efficiently express rCheA and rCheY. The two rabbit antisera and IgGs aginst rCheA and rCheY had 1:4 and 1:2 immunodiffusion titers, respectively. Hydrochloric acid, sulfuric acid and acetic acid were able to induce chemotactic movement of H. pylori. Both rCheA-IgG and closantel sodium with certain concentrations could weaken the chemotactic ability of H. pylori (P<0.05).Conclusion The prokaryotic expression systems of H. pylori cheA and cheY genes were successfully generated in this study. Hydrogen ion (H+) is the inducer for chemotaxis of H. pylori. rCheA-IgG as well as closantel sodium can inhibit H+-induced chemotaxis of H. pylori.