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目的:构建幽门螺杆菌ArsRS双组分系统基因突变株。方法:以幽门螺杆菌标准菌株11637为模板,PCR扩增ArsS、ArsR基因;扩增产物分别克隆于载体pID700A1中,化学法转化感受态大肠杆菌E.coli DH5α,使其在细菌内同源重组,双酶切筛选得到重组载体,然后将重组载体经电转化法转化幽门螺杆菌标准菌株,获得幽门螺杆菌ArsS、ArsR突变株。结果:PCR获得了360bp、350bpArsS、ArsR基因,构建了插入ArsS、ArsR基因的重组载体pID700A1-ArsS、pID700A1-ArsR。经双酶切分析显示,所得条带与设计结果完全一致。PCR方法扩增突变株结果显示ArsS、ArsR基因已缺失。结论:成功构建了幽门螺杆菌ArsS、ArsR突变株,为ArsS、ArsR基因的检测和新型药物靶点以及疫苗的研究奠定基础。
Objective: To construct Helicobacter pylori ArsRS two-component system mutant. Methods: ArsS and ArsR genes were amplified by PCR using the standard strain of Helicobacter pylori 11637 as a template. The amplified product was cloned into vector pID700A1 and transformed into competent E. coli DH5α chemically to make homologous recombination in bacteria , Double-digested to obtain the recombinant vector, and then the recombinant vector was electroporated into Helicobacter pylori standard strains to obtain Helicobacter pylori ArsS, ArsR mutant strains. Results: 360bp, 350bp ArsS and ArsR genes were obtained by PCR. Recombinant plasmids pID700A1-ArsS and pID700A1-ArsR with ArsR gene and ArsR gene were constructed. The double digestion analysis showed that the resulting band and the design results are exactly the same. PCR amplification of mutants results show ArsS, ArsR gene has been deleted. Conclusion: The ArsS and ArsR mutant strains of Helicobacter pylori were successfully constructed, which laid the foundation for the detection of ArsS and ArsR genes and new drug targets and vaccine research.