cag致病岛缺失的中国幽门螺杆菌突变菌株的构建及鉴定

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目的 构建cag致病岛缺失的中国幽门螺杆菌 (H .pylori)突变株。为cag致病岛功能及H .pylori感染致病多样性机制的研究奠定基础。方法 运用基因重组方法将氯霉素抗性基因(CmR)连接到PCR扩增cag致病岛两端区域产生的 2个目的基因片段之间 ,并共同插入到pBluescriptSK( )载体的多克隆位点中 ,构建出带氯霉素抗性标志的缺失突变载体pBS cag mutant,将突变载体转化到含完整cag致病岛基因的突变受体H .pylori菌株中 ,根据同源重组原理 ,筛选出cag致病岛缺失的中国H .pylori突变株 ,并经PCR方法鉴定。结果 构建的突变载体经内切酶酶切分析显示 :产生的条带与设计结果完全一致。PCR方法扩增cagA、cagⅠ、cagⅡ、尿素酶等基因的结果显示 :筛选出的细菌为缺失cag致病岛基因的H .pylori菌株。结论 我们成功地构建出 1株中国人来源的、cag致病岛缺失的H .pylori突变株 ,对于阐明cag致病岛功能及其在H .pylori致病中的地位及作用研究具有重要的价值及意义 Objective To construct a Chinese H. pylori mutant with deletion of cag pathogenicity island. Which laid the foundation for the study of the pathogenic island function of cag and pathogenic diversity of H.pylori infection. Methods The gene encoding chloramphenicol resistance gene (CmR) was inserted into the two target gene fragments generated by PCR amplification of the two ends of cag pathogenicity island and inserted into the multiple cloning site of pBluescriptSK () vector , We constructed the deletion mutation vector pBS cag mutant with chloramphenicol resistance marker and transformed the mutant vector into mutant H. pylori strain with complete cag pathogenicity island gene. According to the homologous recombination principle, cag Pathogenicity island deletion of Chinese H.pylori mutant and identified by PCR method. Results The constructed mutated vector was digested by endonuclease and showed that the bands produced were completely consistent with the design results. PCR amplification of cagA, cag Ⅰ, cag Ⅱ, urease and other genes showed that the selected bacteria were cp. Virulence strains lacking cag pathogenicity island genes. Conclusions We successfully constructed a Chinese-origin mutant strain of H.pylori with cag pathogenicity island deletion, which is of great value in elucidating the function of cag pathogenicity island and its role in the pathogenesis of H.pylori And meaning
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