幽门螺杆菌Ⅳ型分泌系统cagQ基因缺失株的构建与鉴定

发布时间：2018-01-08 10:36

本文关键词：幽门螺杆菌Ⅳ型分泌系统cagQ基因缺失株的构建与鉴定　出处：《江苏大学学报(医学版)》2014年06期 　论文类型：期刊论文

【摘要】：目的:构建幽门螺杆菌(Helicobacter pylori,H.pylori)Ⅳ型分泌系统cag Q基因缺失株。方法:通过PCR扩增出cag Q基因编码区侧翼同源臂序列,经TA克隆后进行双酶切,并将纯化后上下游同源臂连接至两端含有卡那霉素抗生素基因的载体p Bluscript SK II(-),构建为cag Q基因自杀质粒。利用电穿孔法将自杀质粒导入H.pylori,进行同源重组。培养后通过卡那霉素抗生素筛选出基因缺失株,并经PCR及核酸序列分析进一步确定基因缺失株。结果:H.pylori的cag Q基因自杀质粒p Blue KM40-△cag Q经酶切验证无误,进行电转化后经PCR及核酸序列分析结果正确。结论:成功获得H.pylori cag Q基因缺失株,命名为Hp26695-△cag Q。
[Abstract]:Objective: to construct Helicobacter pylori pylori. Methods: the flanking arm sequence of the coding region of cag Q gene was amplified by PCR and digested by double enzyme after TA cloning. After purification, the upstream and downstream homologous arms were ligated to the vector p Bluscript SK II- containing kanamycin antibiotic gene at both ends. The suicide plasmid of cag Q gene was constructed. The suicide plasmid was introduced into H. pylori by electroporation and homologous recombination. After culturing, kanamycin antibiotic was used to screen out the gene deletion strain. Results the cag Q gene suicide plasmid p Blue KM40-cag of H. pylori was further identified by PCR and nucleic acid sequence analysis. Q was confirmed by enzyme digestion. The results of PCR and nucleic acid sequence analysis were correct. Conclusion: H.pylori cag Q gene deletion strain was successfully obtained and named Hp26695- cag Q.
【作者单位】：江苏大学医学院;
【基金】：江苏省自然科学基金青年基金资助项目(BK20130542) 江苏省科技创新与成果转化(生命健康科技)专项基金资助项目(BL2012047) 高等学校博士学科点专项科研基金资助项目(20123227110008)
【分类号】：R378
【正文快照】： 幽门螺杆菌(Helicobacter pylori,H.pylori)是一类微需氧、革兰染色阴性螺杆菌,主要定植于人胃黏膜表面及十二指肠球部[1],全球感染率达50%,慢性感染常导致胃炎,甚至胃十二指肠溃疡、胃癌[2]。H.pylori中含有长度约40 kb的基因片段,即cag致病岛(cytotoxin-associated gene-pat

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