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目的:应用信号标签突变技术(Signature tagged mutagenesis,STM)构建幽门螺杆菌突变体文库重组质粒。方法:采用平末端内切酶随机酶切幽门螺杆菌基因组DNA,并回收300～500bp片段(记作Fr),基因重组技术构建重组质粒pID700-Fr,电转化E.coliDH5α筛选阳性克隆,提取重组质粒酶切鉴定。结果:成功构建了1200个幽门螺杆菌STM文库重组质粒。结论:STM技术可用于幽门螺杆菌致病以及耐药相关基因的筛选,为幽门螺杆菌致病及耐药机理的研究奠定了基础。也将为幽门螺杆菌的治疗提供新的药物靶点。
Objective: To construct a recombinant plasmid of Helicobacter pylori mutant library by using Signature tagged mutagenesis (STM). Methods: Genomic DNA of Helicobacter pylori was randomly digested with blunt endonuclease and 300 ~ 500bp fragment was recovered (denoted as Fr). Recombinant plasmid pID700-Fr was constructed by gene recombination technology and electroporated into E.coli DH5α to screen positive clones for recombination Plasmid digestion identification. Results: 1200 Helicobacter pylori STM library plasmids were successfully constructed. Conclusion: STM can be used in the pathogenesis of Helicobacter pylori and the screening of drug-resistance-related genes, which lays the foundation for the pathogenesis of H. pylori and the mechanism of drug resistance. Will also provide a new drug target for the treatment of Helicobacter pylori.