幽门螺杆菌对甲硝唑耐药相关基因片段的筛选及克隆分析

Screening for metronidazole-resistance associated gene fragments of Helicobacter pylori by suppression subtractive hybridization

目的:筛选幽门螺杆菌(Hp)对甲硝唑耐药相关基因片段。方法:以5株临床分离的耐药株作为被检菌,1株敏感株作为参考菌,采用抑制差减杂交技术进行基因组差异研究,获取Hp对甲硝唑耐药相关的基因片段,并以斑点杂交方法对所获的基因片段进行了杂交鉴定。结果:在获得的差减阳性克隆中,经斑点杂交筛选后共有37个基因片段的拷贝数在耐药和敏感菌株中存在差异(2倍以上),而其中17个片段其拷贝数存在明显差异(3倍以上)。对以上17个差异片段进行单向测序,根据最高同源性比对结果,分别代表二肽ABC载体渗透酶(dppB)、二肽ABC载体结合蛋白(dppA)等10个基因的同源基因片段在耐药株中存在高拷贝数。结论:所获得的10个差异基因片段可能与幽门螺杆菌对甲硝唑耐药相关。

Objective： To screen metronidazole （MTZ） -resistance associated gene fragments of Helicobacter pylori （H. pylori） by suppression subtractive hybridization （SSH）. Methods： The suppression subtractive hybridization （SSH） was used to screen the different DNA fragments between MTZ-resistant and -susceptible clinical strains of H. pylori. The resistant strains specific gene fragments were obtained by SSH and identified by dot blotting. Results： Among the 120 subtractive colonies which were randomly chosen, 37 DNA fragments were different （≥2 times） in DNA-copy numbers between resistant and susceptible strains and 17 of them were significantly different （≥3 times）. These 17 DNA fragments were sequenced subsequently. Ten of them were new sequences and the other 7 were duplicated sequences. These sequences represented respectively ：depeptide ABC transporter periplasmic dipeptide-binding protein （dppA）, permease protein （dppB）,et al. Conclusion： Gene fragments specific to MTZ-resistant H. pylori strains were obtained by SSH and these genes may associated with MTZ-resistance of H. pylori.