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目的基于融合PCR技术(SOE-PCR)、T载体克隆技术,构建结核分枝杆菌(Mycobacterium tuberculosis,MTB)双组份系统缺失突变载体的方法。方法利用SOE-PCR技术,将PhoP、PhoR和PhoPR基因上、下游的同源臂与卡那霉素抗性基因融合,构建PhoP、PhoR和PhoPR突变片段,然后将突变片段直接与T载体连接,将其转入E.coli DH5α感受态细胞并筛选抗性克隆,进而获得MTB PhoP、PhoR和PhoPR缺失突变载体。结果成功构建MTB PhoPR双组份系统缺失突变载体,与传统方法相比,效率高、周期短。结论基于融合PCR技术和T载体克隆技术成功构建MTB PhoPR双组份系统缺失突变载体,为下一步构建MTB突变株以及相关研究奠定基础。
OBJECTIVE: To construct a two-component deletion mutant vector of Mycobacterium tuberculosis (MTB) based on fusion PCR (SOE-PCR) and T vector cloning techniques. Methods SOE-PCR was performed to fuse PhyP, PhoR and PhoPR homology arms with kanamycin resistance genes. PhoP, PhoR and PhoPR mutants were constructed. Then, the mutated fragments were directly linked to T vector, The recombinant plasmid was transformed into E.coli DH5α competent cells and the resistant clones were screened to obtain MTB PhoP, PhoR and PhoPR deletion mutants. Results The MTB PhoPR two-component deletion vector was successfully constructed, which was more efficient and shorter in duration than the traditional method. Conclusion The MTB PhoPR two-component system deletion mutant vector was successfully constructed based on the fusion PCR and T vector cloning technology, which laid the foundation for further construction of MTB mutant strains and related research.