问号钩端螺旋体cola基因产物胶原酶活性及其致病机制研究 赵金方 谈 .doc

问号钩端螺旋体olA基因胶原酶活性及其致病机制研究 Kokouvi Kassegne ［摘要］目的：了解钩端螺旋体colA基因产物胶原酶活性colA基因表达水平变化和产物分泌情况。方法：采用PCR及其产物测序colA基因并了解其序列保守性。构建问号钩体黄疸出血群赖型赖株colA基因原核表达系统，Ni-NTA亲和层析法提取表达的目的重组蛋白rColA。rColA水解I～IV型天然胶原蛋白及Azocoll和Pz-肽合成底物能力并测定其Km和Kcat值。采用实时荧光定量RT-PCR钩体赖株L-02、HEK293细胞colA-mRNA水平变化及ColA分泌情况。结果：不同血清群序列相似性达99.4%100%。colA基因表达系统能有效表达rColA。rColA能不同程度地水解上述6种底物，但水解III型胶原蛋白能力最强（p0.05），其Km和Kcat值分别为2.16 mg/ml和35.6 h-1。问号钩体赖株感染各靶细胞colA-mRNA水平显著升高（p0.01），问号钩体-细胞共培养物上清中可检出ColA。结论：问号钩体colA基因为序列保守、分布广泛的胶原酶编码基因，感染细胞时该基因产物表达上调并外分泌，从而在问号钩体感染宿主过程中发挥实际作用。 ［关键词］问号钩端螺旋体；colA基因；胶原酶；表达；分泌 Collagenase activity and pathogenic mechanism of colA gene product of Leptospira interrogans Zhao Jinfang1, Tan Panli1, Wang Zhejiong1, Hu Weilin2, Yan Jie2, Kokouvi Kassegne2. (1. Department of Laboratory Medicine, the First Afiliated Hospital of Zhejiang Chinese Medical University, Hangzhou 310006, China; 2. Department of Medical Microbiology and Parasitology, Zhejiang University School of Medicine，Hangzhou 310058, China) Corresponding author: Kokouvi Kassegne, Email: med_bp@zju.edu.cn ［Abstract］Objective: colA gene in different serogroups of L. interrogans, and determine the collagenase activity, expression and secretion of colA gene product of the spirochete during infection of host cells. Methods: PCR and its product sequencing were performed to detect the colA gene and sequence conservation of seven L. interrogans serogroups that prevailing in China. A colA gene prokaryotic expression system of L. interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai was generated. Ni-NTA affinity chromatography was used to extract the expressed target recombinant protein rColA. By using spectrophotometry, the ability of rColA to hydrolyze I～IV type natural collagen proteins and two synthetic substrates (Azocoll and Pz-peptide), and the Km and Kcat values were determined. Real-time fluorescent quantitative RT-PCR and Western Blot assay were applied to detect the colA-mRNA level change and ColA secretion of L. inte