为构建结核分枝杆菌毒素-抗毒素系统mazEF6缺失突变株，并对其表型进行初步探讨，首先用聚合酶链反应(PCR)分别从H37Rv标准株和PUC-19K质粒扩增出mazEF6基因的同源臂及卡那霉素抗性基因kan；然后应用融合PCR技术将mazEF6基因的同源臂与kan基因进行杂交拼接，获得目的融合片段，将该融合片段克隆于pMD-19T(simple)载体形成自杀质粒pMD-19T-ΔmazEF6-kan，并将自杀质粒转化至大肠埃希菌DH5α中；最后利用电穿孔技术将自杀质粒电转至H37Rv标准株中，在卡那霉素抗性改良罗氏培养基上筛选H37Rv ΔmazEF6缺失突变株单个菌落，提取阳性菌株全基因组DNA为模板，PCR扩增克隆片段并测序。将所获得的H37Rv ΔmazEF6缺失突变株进行遗传稳定性检测后，对其表型进行初步研究。结果显示，该缺失株在15代内未发生回复性突变；与野生株相比，缺失株生长速度缓慢且细菌形态短小。本研究证实，融合PCR技术便于快速获得结核分枝杆菌缺失突变株；结核分枝杆菌在缺失毒素-抗毒素系统mazEF6基因后生存能力下降，这为进一步研究毒素-抗毒素系统的作用奠定了基础。

To study the toxin-antitoxin system mazEF6 of Mycobacterium tuberculosis (M. tuberculosis), deletion mutants were constructed and subjected to phenotype analysis. First, the flanking (homology arms) of mazEF6 gene from H37Rv and kanamycin resistance gene (kan gene) from plasmid PUC-19K were amplified by polymerase chain reaction (PCR) respectively. Second, fusion PCR was used for the hybrid splicing of mazEF6 homology arms and kan gene, and the desired fusion fragment was obtained. Then the fragment was cloned into pMD-19T (simple) vector to form a suicide plasmid (pMD-19T-ΔmazEF6-kan), and the suicide plasmid was transformed into Escherichia coli (E. coli) DH5α. At last, the constructed plasmid was transformed into H37Rv by electroporation. Single colonies of M. tuberculosis were screened on L-J medium with kanamycin, the genomic DNA of positive strains were extracted, and the targeted fragments were amplified by PCR and sequenced. The genetic stability and other phenotypes of the H37Rv ΔmazEF6 deletion mutant were studied. The results showed that the deletion mutant strains did not present reverse mutant within 15 generations. Compared with the wild-type strains, H37Rv ΔmazEF6 deletion mutant strains grew more slowly and the bacterial cell was relatively shorter. This study demonstrated that it is practical to obtain M. tuberculosis deletion mutant by fusion PCR technology, and the survival ability of M. tuberculosis without toxin-antitoxin mazEF6 gene is decreased.