幽门螺杆菌hp0596基因缺失突变体的构建

Construction of an hp0596 gene deletion mutant of Helicobacter pylori

目的:构建幽门螺杆菌(Helicobacter pylori,H.pylori)hp0596基因缺失突变体,为进行hp0596基因功能研究奠定基础.方法:利用PCR技术,从H.pylori 26695基因组分别扩增得到hp0596基因的上游同源臂和下游同源臂,与两端带有FRT位点的氯霉素抗性基因片段共同构建同源重组载体;以重组载体为模板扩增打靶片段,将其转化入H.pylori 26695;在抗生素选择压力下,打靶片段和菌体基因组发生同源重组,通过氯霉素抗性筛选得到带有抗性标记的重组菌.结果:构建的突变载体经限制性内切酶酶切分析显示,产生的条带与预计结果完全一致.PCR方法扩增突变株0596,cmr基因显示,0596基因已经被完全敲除,经DNA测序,RNA水平,蛋白质水平证实筛选得到了0596基因缺失的H.pylori26695△0596突变株.连续培养7代后,H.pylori26695△0596突变株具有良好的稳定性.结论:获得了H.pylori26695△0596基因缺失突变体.

AIM： To construct an hp0596 gene deletion mutant of Helicobacter pylori （H.pylori） to provide a basis for further study of the functions of the hp0596 gene.METHODS： Two homologous arms upstream and downstream of the hp0596 gene were amplified from H.pylori 26695 genomic DNA and cloned into a pBluescript SK II（-） vector carrying a chloramphenicol resistance cassette flanked by two FRT sites to construct a recombinant vector.The target fragment was then amplified from the recombinant vector and transformed into H.pylori 26695.Under antibiotic selective pressure,homologous recombination occurred between the target fragment and the genome of host strain.The recombinants were selected on chloramphenicol agar plates and identified by PCR.RESULTS： Restriction endonuclease analyses showed that the recombinant vector （pBs-0596） was successfully constructed.An hp0596 gene deletion mutant of H.pylori 26695 was successfully obtained after identification by PCR,direct sequencing,resistance analysis,and detection of hp0596 expression at RNA and protein levels.After culturing for 7 generations,it was confirmed that the hp0596 gene deletion mutant of H.pylori 26695 was stable.CONCLUSION： An hp0596 gene deletion mutant of H.pylori 26695 has been obtained successfully.