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目的构建鼠疫耶尔森菌(鼠疫菌)中基因组无痕修饰的技术平台,深入研究鼠疫菌相关基因的功能及作用机制。方法通过不对称PCR扩增抗性盒片段(含修饰靶标区域上下游同源臂),将其导入含pKD46质粒的鼠疫菌中。在阿拉伯糖的诱导下,pKD46质粒可表达与同源重组相关的3个酶(Exo,Beta和Gam蛋白),诱导重组后再导入p KSI-1质粒与目的基因的重组载体,与抗性盒进行第二次同源重组。通过加入阿拉伯糖和IPTG诱导pREDTKI表达重组酶和I-SceⅠ酶,可对未发生重组的抗性盒片段和p KSI-1质粒上的I-SceⅠ位点酶切,从而起到消除抗性盒与质粒作用,达到无痕修饰的目的。结果与结论成功构建了鼠疫菌的Δwaa A和waa A(△9nt)两个突变株。根据不同的实验目的选择相应的基因敲除方法,得到的无痕修饰菌株为鼠疫菌相关基因的功能研究奠定了基础。
OBJECTIVE: To construct a genome-free technique platform for Yersinia pestis (Yersinia pestis) and to study the function and mechanism of Yersinia pestis-associated genes. Methods The resistance cassette fragments (including the upstream and downstream homology arms of the modified target region) were amplified by asymmetric PCR and introduced into the plague bacterium containing pKD46 plasmid. Under the induction of arabinose, the pKD46 plasmid can express three enzymes (Exo, Beta and Gam proteins) related to homologous recombination, induce recombination, and then introduce the recombinant vector of p KSI-1 plasmid and the target gene, The second homologous recombination. The pREDTKI-expressing recombinase and I-SceI enzyme are induced by the addition of arabinose and IPTG to cleave the I-SceI site on the non-recombined resistance cassette fragment and p KSI-1 plasmid to thereby eliminate the resistance cassette With the role of plasmid, to achieve the purpose of seamless modification. Results and Conclusion Two mutant strains Δwaa A and waa A (△ 9nt) were successfully constructed. According to different experimental purposes, the corresponding gene knockout method is selected, and the resulting non-modified modified strain lays the foundation for the functional study of the related gene of Y. pestis.