Use of a flexible cassette method to generate a double unmarked Mycobacterium tuberculosis tlyA plcABC mutant by gene replacement
Use of a flexible cassette method to generate a double unmarked Mycobacterium tuberculosis tlyA plcABC mutant by gene replacement
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DOI：
10.1099/00221287-146-8-1969
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发表时间：
2000-08-01
期刊：
影响因子：
2.8
通讯作者：
Stoker, NG
中科院分区：
文献类型：
--
作者：
Parish, T;Stoker, NG
分枝杆菌研究领域的进展受到阻碍，因为不能容易地产生生长缓慢的分枝杆菌的确定的突变菌株以研究特定基因的功能。描述了一种有效的方法，该方法已用于产生几种突变体，包括结核分枝杆菌的第一个双未标记缺失菌株。构建了四种突变体：编码三种磷脂酶C的plcABC簇的标记缺失; plcABC和tlyA（编码溶血素）中单独的未标记缺失;以及双未标记突变体tlyA Delta plcABC Delta。为了实现这一点，设计了两个系列的载体，其中第一个称为pNIL，允许在各种方便的限制性位点处操纵靶基因序列。第二个系列，命名为pGOAL，包含侧翼为Pad限制酶位点的标记盒。然后通过将来自pGOAL载体的标记盒克隆到具有修饰的感兴趣基因的pNIL载体的单个PacI位点中，获得最终的自杀质粒载体。最后，采用两步策略，即首先选择单交换事件，然后进行第二次交换的筛选以产生突变菌株。这种技术现在将允许构建潜在的疫苗株，而不包括抗生素抗性标记，能够产生多个定义的突变，并可能产生更微妙的定义突变，如点突变。
Progress in the field of mycobacterial research has been hindered by the inability to readily generate defined mutant strains of the slow-growing inability to readily generate defined mutant strains of the slow-growing mycobacteria to investigate the function of specific genes. An efficient method is described that has been used to generate several mutants, including the first double unmarked deletion strain of Mycobacterium tuberculosis. Four mutants were constructed: a marked deletion of the plcABC cluster, which encodes three phospholipases C; separate unmarked deletions in plcABC and tlyA (encoding a haemolysin); and a double unmarked mutant tlyA Delta plcABC Delta. To accomplish this, two series of vectors were designed, the first of which, named pNIL, allows manipulation of the target gene sequence at a variety of convenient restriction sites. The second series, named pGOAL, contains marker cassettes flanked by Pad restriction enzyme sites. The final suicide plasmid vectors were then obtained by cloning a marker cassette from a pGOAL vector into the single PacI site of the pNIL vector with the modified gene of interest. Finally, a two-step strategy was employed whereby single cross-over events were first selected, then screening for the second cross-over was carried out to yield the mutant strains. This technique will now allow the construction of potential vaccine strains without the inclusion of antibiotic resistance markers, the ability to make multiple defined mutations and the possibility of making more subtle defined mutations, such as point mutations.