| Among world population, 50% is infected with Helicobacter pylori (H.pylori) at present. The infected population of China is 50 ~ 70%, which is higher than the world average rate. Therefore, the anti-Helicobacter pylori infection control and treatment is an important medical and social issue we are facing with. With the increasing use of H.pylori eradication therapy antibiotics in the world, antibiotics-resistant H.pylori is prominent problem. The emergence of H.pylori drug-resistance is related to the antibiotic-related gene mutation. The molecular mechanism of H.pylori drug-resistance have been widely studied. It was demonstrated that clarithromycin-resistance is associated with two point mutations (A2142G , A2143G) in 23S rRNA of the H.pylori ,Metronidazole-resistance is associated with mutation of rdxA and rdxA gene encoding nitroreductase which can restore the generation of bactericidal activity with the metabolic products. So each drug has its own resistant gene-determining region. The point mutations and insertion loss of expression in these regions may cause abnormal changes in the structure and function of expressed proteins, resulting in the emergence of drug resistance. However,drug-resistance related genes and their mechanisms can not be fully found out by this method. Thus the new method of drug resistance analysis and drug target discovery, is the urgent need to address the important issue.Signature-tagged mutagenesis study of microbial pathogens is the most widely used methods in recent years. Herein, the gene in question is inactivated by insertional mutation; a transposon is used which inserts itself into the gene sequence. When that gene is transcribed and translated into a protein, the insertion of the transposon affects the protein structure and prevents it from functioning. In STM, mutants are created by random transposon insertion and each transposon contains a different 'tag' sequence that uniquely identifies it. If an insertional mutant bacterium exhibits a phenotype of interest , such as susceptability to an antibiotic it was previously resistant to, then we will sequence its genome and run a search for any of the tags used in the experiment. When a tag is located, the gene that it disrupts is also thus located. At present, the application of this technology has been applied in more than 10 kinds of pathogenic microorganisms for virulence factors discovery. It also has been proved to be an effective way to resistant gene screening and a variety of specific functions in related genes.Two-component system is in the most of bacteria .Its sophisticated signal transduction system histidine protein kinase (HPK) and the effect of regulatory protein (RR) composed of two components, the fuction is adapt to the change of environment and to generate appropriate cell reaction, Study shows that, H pylori ArsRS two-component system is the most important reasons why H pylori can colonize in gastric mucosal in a long-term . Therefore, we believe that ArsRS two-component system may be associated with H pylori resistance to some extent. In the two-component system,ArsS and ArsR regulate the majority of H pylori acid resistance genes, ureABEFGHI,CA,NiKR,NixA,hypB,hypA,HspA,Fur,aspA;dynamic gene,flaABEGH and virulence gene cagA . These genes are the key of H pylori growth,pathogenicity and drug resistance.Study the regulation System of these genes will help us to further understand the pathogenicity and the mechanism of resistance of H pylori.In this study we firstly choose the main control system , ArsRS two-component system of H.pylori, as the mutation target. The fragments of ArsR and ArsS were amplified using PCR from total DNA of standard H.pylori strains,and then cloned into vector pID700A1 containing chloramphenicol-resistant gene by transforming into E.coli DH5α. After identification by restriction enzyme digestion and DNA sequencing,the correct recombinant vectors pID700A1-ArsS and pID700A1-ArsR were electrotransfected into standard H.pylori strain. Finally, the chloramphenicol -resistant clones were collected, and gene mutations were detected using PCR. We found that the growth rates of ArsS and ArsR deleted mutants were significant lower than that of wild type. To our surprise, the mutants also showed sensitive to clarithromycin and metronidazole. The results verified that the growth activity of H.pylori was close with ArsRS, and for the first time indicated that ArsRS was associated with drug sensitivity.Using this vector-based but not transposon-based STM method, we digested total DNA of Helicobacter pylori with four restriction enzyme, and recovered the 300-500bp DNA fragments after electrophoresis. After cloning these fragments into the vector and transferring into the standard strain of H.pylori, we obtained 70 mutants with chloramphenicol-resistance. This work lays the foundation for further large-scale construction of mutant library and identification of drug-resistance associated genes of H.pylori.
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