| Objective:Helicobacter pylori is a micro-aerophilic, Gram-negative bacterium that can efficiently colonizes the gastric epithelial cells or gastric mucin. It is estimated that H.pylori infects at least half of the world's population. The chronic, persistent infection by H.pylori may cause many gastric diseases including chronic atrophic gastritis, peptic ulcers, mucosa-associated lymphoid tissue (MALT) lymphoma and gastric cancer. H.pylori is equipped with numerous virulence factors, which facilitate the colonization and damage of gastric mucosa. Some of the virulence factors have been identified such as CagA, VacA and UreB, while many other factors that maybe important in the pathogenesis are still unknown. The aim of this study was to identify novel gastric disease-related antigens using in vivo knock-out approach from H.pylori and characterize their role in the pathogenesis as well.Methods and result:In section one, cagA and hp0596 were selected as the target genes for in vivo gene knock-out in H.pylori. Upstream and downstream homologous fragments of the target genes from H.pylori26695 as well as chloramphenicol resistance cassette flanked with two FRT sites were amplified, then all the fragments were inserted into plasmid pBlueskript skⅡ(+) to construct a recombinant vector. The target cassette then amplified from the recombinant vector and transformed into H.pylori26695. Under the pressure of antibiotic selection, homologous recombination occurred between the cassette and H.pylori chromosome, and the final recombinants were selected on chloramphenicol agar plate. The mutant strains were verified by colony PCR. Some basic factors were optimized, such as temperature-sensitive replication vector, length of homologous regions, high-efficiency electroporation protocol, antibiotic concentration, and the method for identification of positive recombinant. By using this procedure, two candidate genes cagA and hp0596 were knocked out from the chromosome. The mutant strains were verified by colony PCR, RT-PCR, total protein SDS-PAGE and western blot. All the results demonstrated that cagA and hp0596 gene was successfully deleted from H.pylori chromosome.In section two, using the sacB gene of Bacillus subtilis, we developed a sucrose-based counterselection system that allows the introduction of unmarked mutations into H.pylori. A comprehensive phenotypic analysis of the mutants was explored subsequently. Specifically, we obtained urease protect and assist its colonization played an important role in regard to a key gene responsible for in H.pylori through bioinformatic analysis. According to homologous recombination, ureB was deleted successfully. Combined with counterselection system the markerless mutant was gained, which named H.pylori26695△ureB and urease enzyme activity had been lost. This result raised the possibility that we could exploit the sucrose sensitivity phenotype conferred by sacB expression to perform a functional exchange of ureB alleles.In the third part of this work, we construct a E. coli-H. pylori shuttle vector in order to achieve expression of heterogenous intestinal adhesion protein on outer membrane of HPSS1. We use theλRed recombination system to reform the shuttle plasmid pHH43, in brief, the bacterial ghosts gene GenE together with the temperature-sensitive promoter and the chloramphenicol resistance gene of pHH43 were substituted with Salmonella adhesin operon lpfABCDE and kanamycin resistance gene by homologous recombination. The reconstructive plasmid was transformed into HPSS1, and then the expresson of adhesin lpfA from the outer membrane proteins of H.pylori was verified.Conclusions:Our work explore a mutagenesis procedure designed for gene replacement in H. pylori, the results indicate that the in vivo recombination is a useful method for function analysis of H.pylori protein. A E. coli-H. pylori shuttle vector was also constructed and a heterogenous protein was successfully expressed.
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