中文摘要：

目的了解问号钩端螺旋体（简称钩体）鞘磷脂酶类溶血素基因sph1—sph4产物溶血活性及其感染细胞后转录水平的变化。方法以致病性问号钩体黄疸出血群赖型赖株、波摩那群波摩那型罗株和非致病性双曲钩体三宝垄群Patoc型PatocⅠ株基因组DNA为模板，采用PCR扩增全长sph1-sph4基因片段，扩增产物T—A克隆后测序。构建sph1—sph4基因原核表达系统，采用SDS-PAGE检测目的重组蛋白rSph1—rSph4的表达情况，Ni—NTA亲和层析柱提纯rSph1—rSph4。采用绵羊血平板对rSph1～rSph4溶血活性进行鉴定，实时荧光定量RT—PCR检测问号钩体赖株感染J774A.1细胞前后sph1～sph4基因转录水平的变化。结果问号钩体赖株和罗株基因组DNA中均能扩增sph1～sph4基因，双曲钩体PatocⅠ株则否。与报道的相应基因序列比较，所克隆的sph1—sph4基因核苷酸序列相似性均为100％。所构建的原核表达系统能分别表达目的重组蛋白rsph1～rSph4。rSph1～rSph4均有溶血活性，其中以rSph2溶血活性最强。问号钩体赖株感染J774A．1细胞后，sph1—sph4基因转录水平均上调，其中sph2和sph4基因mRNA水平上调更为明显。结论sph1—sph4基因仅存在于致病性问号钩体中，其表达产物有溶血活性。问号钩体赖株感染细胞后sph1～sph4基因转录水平的上调，提示此类鞘磷脂酶类溶血素可能在问号钩体感染宿主过程中有重要作用。

英文摘要：

Objective To determine the hemolytic activity of products of sphingomyelinase hemolysin encoding genes of Leptospira interrogans, and the transcriptional level alterations in the infected host cells. Methods By using genomic DNAs of pathogenic L. interrogans serovar serogroup Icterohaemorrhagiae serovar Lai strain Lai and serogroup Pomona serovar Pomona strain Luo, and non-pathogenic L. biflexa serogroup Samaranga serovar Patoc strain Patoc Ⅰ as templates, PCRs were performed to amplify entire sph1-sph4 genes. The amplified products were sequenced after T-A cloning. Prokaryotic expression systems of sph1-sph4 genes were respectively constructed, and the expressions of target recombinant proteins rSph1-rSph4 were examined by SDS- PAGE. Ni-NTA affinity chromatographic column was used to extract the expressed rSph1-rSph4. Hemolytic activities of rSph1-rSph4 on sheep blood agar plate were identified. Transcription alterations of sph1-sph4 genes in L. interrogans strain Lai after infected J774A. 1 cells were measured by real-time fluorescence quantitative RT- PCR. Results From genomie DNAs of both L. interrogans strain Lai and Luo, but not from that of L. biflexa strain Patoc Ⅰ , the target fragments of sph1 -sph4 genes could be amplified. All the cloned sph1-sph4 genes had 100% nucleotide sequence identities compared to the corresponding reported sequences. The constructed prokaryotic expression systems were able to efficiently express the target recombinant proteins rSph1-rSph4, respectively. All the rSph1-rSph4 had hemolytic activities, and among the four products rSph2 displayed the strongest hemolytic activity. After L. iraerrogans strain Lai infecting J774A. 1 cells, the transcriptional levels of sph1-sph4 genes were remarkably up-regulated, especially for mRNA levels of sph2 and sph4 genes. Conclusion sph1- sph4 genes exist only in pathogenic L. interrogans species, and their products have hemolytic activity. The upregulation of sph1-sph4 gene transcriptional levels in L. interrogans strain Lai after infect