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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.
OBJECTIVE: To construct the hp0596 deletion mutant of Helicobacter pylori (H.pylori), which will lay the foundation for the study of hp0596 gene function.Methods: The upstream of hp0596 gene was amplified by PCR from H.pylori 26695 genome A homologous recombination vector was constructed with the chloramphenicol resistance gene fragment with the FRT site at both ends, the target vector was amplified with the recombinant vector and transformed into H. pylori 26695; Under the selection pressure of antibiotics, homologous recombination occurred between the target fragment and the bacterial genome, and the recombinant bacteria with resistance markers were screened by chloramphenicol resistance.Results: The constructed mutant vector was confirmed by restriction enzyme digestion analysis , And the bands produced were completely consistent with the expected results.PCR amplification of mutant 0596, cmr gene, 0596 gene has been completely knocked out, confirmed by DNA sequencing, RNA level, protein level screening confirmed 0596 gene deletion. pylori26695Δ 0596 mutant.After 7 generations of continuous culture, the H.pylori26695Δ 0596 mutant had good stability.Conclusion: The H.pylori26695Δ 0596 gene deletion mutant was obtained.