Prokaryotic expression of Helicobacter pylori cheA and cheY genes and correlation among the expressed products and bacterial chemotactic behavior / 中华微生物学和免疫学杂志

ABSTRACT

Objective To clone the cheA and cheY genes of Helicobacter pylori for construction of their prokaryotic expression systems, and to establish chemotactic model in vitro of H. pylori for determing chemotaxis-inducing substances and to understand the effects of specific antibody and closantel on inhibiting chemotactic behavior of the microbe. Methods The segments of entire cheA and cheY genes were amplified by PGR and then sequenced after T-A cloning. Prokaryotic expression systems of the genes were subsequent-ly constructed. SDS-PAGE plus Bio-Rad 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 anti-sera 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-lgG and closantel sodium on blocking the bacterial chemotactic behavior were also observed. Results The segments with expected sizes of cheA and cheY genes were obtained by PCR, 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 IgG 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. py-lori. rCheA-IgG, as well as closantel sodium can inhibit H~+-induced chemotaxis of H. pylori.