CAJ | 학술논문

为构建结核分枝杆菌毒素‐抗毒素系统 m azEF6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 m azEF6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。
위구건결핵분지간균독소‐항독소계통 m azEF6결실돌변주，병대기표형진행초보탐토，수선용취합매련반응（PCR）분별종H37Rv표준주화PUC‐19K질립확증출 mazEF6기인적동원비급잡나매소항성기인kan ；연후응용융합PCR기술장 mazEF6기인적동원비여 kan기인진행잡교병접，획득목적융합편단，장해융합편단극륭우pMD‐19T（simple）재체형성자살질립pMD‐19T‐ΔmazEF6‐kan ，병장자살질립전화지대장애희균DH5α중；최후이용전천공기술장자살질립전전지H37Rv표준주중，재잡나매소항성개량라씨배양기상사선H37Rv ΔmazEF6결실돌변주단개균락，제취양성균주전기인조DNA위모판，PCR확증극륭편단병측서。장소획득적H37Rv ΔmazEF6결실돌변주진행유전은정성검측후，대기표형진행초보연구。결과현시，해결실주재15대내미발생회복성돌변；여야생주상비，결실주생장속도완만차세균형태단소。본연구증실，융합PCR기술편우쾌속획득결핵분지간균결실돌변주；결핵분지간균재결실독소‐항독소계통 m azEF6기인후생존능력하강，저위진일보연구독소‐항독소계통적작용전정료기출。
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 H 37RvΔmazEF6 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 .