Muir S, Koopman M B, Libby S J, Joens L A, Heffron F, Kusters J G
Department of Molecular Biology, Research Institute of Scripps Clinic, La Jolla, California 92037.
Infect Immun. 1992 Feb;60(2):529-35. doi: 10.1128/iai.60.2.529-535.1992.
Serpula (Treponema) hyodysenteriae, the etiologic agent of swine dysentery, produces a hemolysin which is thought to be an important factor in the pathogenesis of the disease. We report the cloning, sequencing, and expression of a hemolysin gene (tly) from S. hyodysenteriae B204. A pUC19 gene bank of strain B204 was constructed in the Escherichia coli K-12 strain DH5 alpha, and hemolytic recombinants were identified by plating the library on blood agar plates. From the hemolytic recombinants, a 1.5-kb DNA fragment could be isolated that contained information necessary for the production of a hemolysin/cytotoxin in E. coli. Nucleotide sequence determination of this 1.5-kb fragment showed that it contained an open reading frame capable of encoding a 26.9-kDa protein. The recombinant hemolysin was easily released from E. coli by osmotic shock. As with the native hemolysin, the recombinant hemolysin is EDTA insensitive, thermolabile, and cytotoxic for several eukaryotic cell lines. Southern blot hybridization showed that the cloned S. hyodysenteriae hemolysin gene tly is present in all pathogenic strains of S. hyodysenteriae tested and absent in the nonpathogenic, weakly hemolytic spirochete S. innocens.
猪痢疾密螺旋体（蛇形螺旋体属）是猪痢疾的病原体，可产生一种溶血素，该溶血素被认为是该病发病机制中的一个重要因素。我们报道了来自猪痢疾密螺旋体B204的溶血素基因（tly）的克隆、测序及表达。在大肠杆菌K-12菌株DH5α中构建了菌株B204的pUC19基因文库，并通过将文库接种在血琼脂平板上鉴定溶血重组体。从溶血重组体中可分离出一个1.5kb的DNA片段，该片段包含在大肠杆菌中产生溶血素/细胞毒素所需的信息。对这个1.5kb片段的核苷酸序列测定表明，它含有一个能够编码26.9kDa蛋白质的开放阅读框。重组溶血素很容易通过渗透休克从大肠杆菌中释放出来。与天然溶血素一样，重组溶血素对EDTA不敏感、对热不稳定，并且对几种真核细胞系具有细胞毒性。Southern印迹杂交表明，克隆的猪痢疾密螺旋体溶血素基因tly存在于所有测试的猪痢疾密螺旋体致病菌株中，而在非致病性、弱溶血的螺旋体无害密螺旋体中不存在。
