Stibitz S
Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892, USA.
Gene. 1998 Feb 27;208(2):183-9. doi: 10.1016/s0378-1119(97)00643-4.
A plasmid vector was designed, constructed, and used for the repeated retrieval of the bvgA gene from a number of Bordetella pertussis strains that, due to mutations in this gene, exhibited interesting phenotypes regarding the regulation of virulence genes. The vector was used in a scheme called allelic retrieval that exploits two cross-overs between cloned plasmid and native chromosomal sequences flanking the bvgA gene. This scheme is very similar to allelic exchange through the use of plasmid suicide vectors, but in the case presented here, the non-replicating plasmid that has received the chromosomal gene is recovered, rather than being allowed to be lost due to segregation. Incorporation of the counterselectable sacB gene of Bacillus subtilis in place of the plasmid copy of bvgA allows selection, after recovery in Escherichia coli, for only those plasmids that have retrieved the chromosomal bvgA gene. The validity of this approach was demonstrated by the retrieval of bvgA alleles with distinctive physical markers, as well as by the reintroduction of retrieved bvgA alleles to demonstrate that they conferred the expected phenotypes. It is expected that this approach will be applicable to the analysis of other genes in other bacterial species.
设计、构建了一种质粒载体，并用于从多个百日咳博德特氏菌菌株中反复获取bvgA基因。这些菌株由于该基因发生突变，在毒力基因调控方面表现出有趣的表型。该载体用于一种称为等位基因获取的方案，该方案利用克隆质粒与bvgA基因侧翼的天然染色体序列之间的两次交叉。此方案与通过使用质粒自杀载体进行等位基因交换非常相似，但在本文所述的情况下，已获得染色体基因的非复制性质粒被回收，而不是因分离而丢失。用枯草芽孢杆菌的反选择sacB基因取代bvgA的质粒拷贝，使得在大肠杆菌中回收后，仅能选择那些已获取染色体bvgA基因的质粒。通过获取具有独特物理标记的bvgA等位基因，以及通过重新引入获取的bvgA等位基因以证明它们赋予预期表型，证明了该方法的有效性。预计该方法将适用于分析其他细菌物种中的其他基因。
