Drazek E S, Houng H S, Crawford R M, Hadfield T L, Hoover D L, Warren R L
Department of Infectious and Parasitic Diseases, Armed Forces Institute of Pathology, Washington, D.C. 20306-6000, USA.
Infect Immun. 1995 Sep;63(9):3297-301. doi: 10.1128/iai.63.9.3297-3301.1995.
We constructed a defined purine-auxotrophic mutant of Brucella melitensis 16M by chromosomal gene replacement. We electroporated B. melitensis 16M with suicide plasmids containing a kanamycin resistance cassette that replaced 226 bp at the carboxyl end of purE, the intergenic region, and 18 bases of the purK open reading frame. Recombinant B. melitensis delta purE201 required exogenous purines for growth on minimal media. Purine auxotrophy was complemented by electroporation of B. melitensis delta purE201 failed to grow in human monocyte-derived macrophages, while the growth of wild-type 16M and the complemented strain, delta purE201 (pSD5), increased by nearly two logs. These results suggest that B. melitensis delta purE201 will be attenuated in animals and humans and thus may be useful as a live attenuated vaccine.
我们通过染色体基因替换构建了一株明确的布鲁氏菌16M嘌呤营养缺陷型突变体。我们用含有卡那霉素抗性盒的自杀质粒对布鲁氏菌16M进行电穿孔，该抗性盒替换了purE羧基端226 bp、基因间区域以及purK开放阅读框的18个碱基。重组布鲁氏菌ΔpurE201在基本培养基上生长需要外源性嘌呤。嘌呤营养缺陷通过电穿孔进行互补，布鲁氏菌ΔpurE201在人单核细胞衍生的巨噬细胞中无法生长，而野生型16M和互补菌株ΔpurE201（pSD5）的生长增加了近两个对数级。这些结果表明，布鲁氏菌ΔpurE201在动物和人类中将会减毒，因此可能作为一种减毒活疫苗有用。
