结核分枝杆菌国际标准强毒株H37Rv菌株Pup-蛋白酶体系统Pup基因、Mpa基因、Dop基因和PafA基因缺失突变株的构建

Construction of deletion mutants of the Pup,Mpa,Dop,and PafA genes of the Pup-proteasome system of Mycobacterium tuberculosis using the H37Rv strain

目的利用SOE-PCR技术和T载体技术,快速构建结核分枝杆菌国际标准强毒株H37Rv菌株Pup-蛋白酶体系统Pup基因、Mpa基因、Dop基因和PafA基因缺失突变株,为进一步研究结核分枝杆菌Pup-蛋白酶体系统的功能奠定基础。方法基于同源重组原理,利用SOE-PCR技术和T载体技术分别将各待缺失基因的上下游同源臂与卡那霉素抗性基因融合,构建各基因缺失突变盒;将各基因缺失突变盒与T-Vector pMD 19(Simple)相连接,构建各基因缺失突变自杀载体;利用电穿孔技术将各载体转入H37Rv菌株,通过筛选阳性克隆、菌落PCR鉴定、DNA测序及遗传稳定性检测,获得各目的基因的缺失突变株。结果成功构建了带有1 601bp的特异性置换片段Pup-N-K和1 591bp的特异性置换片段K-Pup-C的Pup基因缺失突变株H37Rv△Pup,带有1 604bp的特异性置换片段Mpa-N-K和1 602bp的特异性置换片段K-Mpa-C的Mpa基因缺失突变株H37Rv△Mpa,带有1 515bp的特异性置换片段Dop-N-K和1509bp的特异性置换片段K-Dop-C的Dop基因缺失突变株H37Rv△Dop及带有1 590bp的特异性置换片段PafA-N-K和1 584bp的特异性置换片段K-PafA-C的PafA基因缺失突变株H37Rv△PafA,且各突变株均具有良好的遗传稳定性。结论利用SOE-PCR技术和T载体技术可以快速构建结核分枝杆菌基因缺失突变株,依据此方法成功构建了H37Rv菌株Pup-蛋白酶体系统Pup基因、Mpa基因、Dop基因和PafA基因缺失突变株,为进一步研究结核分枝杆菌Pup-蛋白酶体系统的功能奠定了基础。

Objective To rapidly construct deletion mutants of the Pup,Mpa,Dop,and PafA genes of the Pup-proteasome system in Mycobacterium tuberculosis using use SOE-PCR and T vector technology and the H37 Rv strain of M.tuberculosis in order to lay the groundwork for study of the Pup-proteasome system of M.tuberculosis. Methods Based on the principle of homologous recombination,homologous arms upstream and downstream of each deleted gene were fused with the anti-kanamycin gene to construct a set of gene deletion mutants using SOE-PCR and T vector technology.Gene deletion mutants were ligated into the pMD19T（Simple）vector to construct a suicide vector for each gene deletion mutant.Each suicide vector was electroporated into the H37 Rv strain.Screening for positive clones,PCR identification,DNA sequencing,and genetic stability testing yielded a deletion mutant of each target gene. Results The deletion mutant H37Rv△Pup was successfully constructed with a specific fragment of 1 601 bp replacing Pup-N-K and a specific fragment of 1 591 bp replacing K-Pup-C.The deletion mutant H37Rv△Mpa was successfully constructed with a specific frag-ment of 1 604 bp replacing Mpa-N-K and a specific fragment of 1 602 bp replacing K-Mpa-C.The deletion mutant H37Rv△Dop was successfully constructed with a specific fragment of 1 515 bp replacing Dop-N-K and a specific fragment of 1509 bp replacing K-Dop-C.The deletion mutant H37Rv△PafA was successfully constructed with a specific fragment of 1590 bp replacing PafA-N-K and a specific fragment of 1 584 bp replacing K-PafA-C.Each deletion mutant exhibited good genetic stability. Conclusion A new technique based on SOE-PCR and T vector technology allowed the rapid construction of deletion mutants of M.tuberculosis.Deletion mutants of the Pup,Mpa,Dop,and PafA genes of the Pup-proteasome system of M.tuberculosis have laid the groundwork for the functional study of the Pup-proteasome system.