Kupsch E M, Aubel D, Gibbs C P, Kahrs A F, Rudel T, Meyer T F
Max-Planck-Institut für Biologie, Abteilung Infektionsbiologie, Tübingen, Germany.
Mol Gen Genet. 1996 Mar 20;250(5):558-69. doi: 10.1007/BF02174444.
A versatile shuttle system has been developed for genetic complementation with cloned genes of transformable and non-transformable Neisseria mutants. By random insertion of a selectable marker into the conjugative Neisseria plasmid ptetM25.2, a site within this plasmid was identified that is compatible with plasmid replication and with conjugative transfer of plasmid. Regions flanking the permissive insertion site of ptetM25.2 were cloned in Escherichia coli and served as a basis for the construction of the Hermes vectors. Hermes vectors are composed of an E. coli replicon that does not support autonomous replication in Neisseria, e.g. ColE1, p15A, or ori(fd), fused with a shuttle consisting of a selectable marker and a multiple cloning site flanked by the integration region of ptetM25.2. Complementation of a non-transformable Neisseria strain involves a three-step process: (i) insertion of the desired gene into a +Hermes vector; (ii) transformation of Hermes into a Neisseria strain containing ptetM25.2 to create a hybrid ptetM25.2 via gene replacement by the Hermes shuttle cassette; and (iii) conjugative transfer of the hybrid ptetM25.2 into the final Neisseria recipient. Several applications for the genetic manipulation of pathogenic Neisseriae are described.
已开发出一种通用穿梭系统，用于与可转化和不可转化的奈瑟氏菌突变体的克隆基因进行遗传互补。通过将一个可选择标记随机插入接合性奈瑟氏菌质粒ptetM25.2中，确定了该质粒内一个与质粒复制及质粒接合转移相容的位点。ptetM25.2允许插入位点两侧的区域在大肠杆菌中进行克隆，并作为构建赫耳墨斯载体的基础。赫耳墨斯载体由一个在奈瑟氏菌中不支持自主复制的大肠杆菌复制子组成，例如ColE1、p15A或ori(fd)，与一个由可选择标记和一个多克隆位点组成的穿梭序列融合，该多克隆位点两侧为ptetM25.2的整合区域。对不可转化的奈瑟氏菌菌株进行互补涉及一个三步过程：(i) 将所需基因插入一个 +赫耳墨斯载体；(ii) 将赫耳墨斯载体转化到含有ptetM25.2的奈瑟氏菌菌株中，通过赫耳墨斯穿梭盒进行基因替换来创建一个杂交ptetM25.2；以及(iii) 将杂交ptetM25.2接合转移到最终的奈瑟氏菌受体中。文中描述了对致病性奈瑟氏菌进行基因操作的几种应用。
