问号钩端螺旋体胶原酶体内和体外的活性研究

In vivo and in vitro activities of Leptospira interrogans collagenase

目的通过检测致病性钩端螺旋体(钩体)胶原酶在体内、外的活性,探讨问号钩体黄疸出血群赖型赖株(赖株)假定的胶原酶(LA0872)在钩体病出血中的可能作用。方法在大肠杆菌中克隆、表达重组赖株la0872,并行相关鉴定及蛋白酶学活性检测。比较不同毒力菌株赖株、赖株减毒株和双曲钩体三宝垄群montevalerio型Monte Valerio株钩体胶原酶的基因序列特异性及转录和酶活水平的差异。测定豚鼠和沙鼠赖株感染模型的血清胶原酶活性水平变化。结果成功构建的重组质粒经酶切和测序鉴定显示位点连接正确,插入序列与GenBank公布的la0872序列完全一致,并证实其具有胶原酶活性;不同毒力菌株中的钩体胶原酶基因序列一致,转录和酶活水平均无显著性差异;豚鼠和沙鼠感染赖株后,宿主体内血清胶原酶活性水平与未感染赖株动物比较差异无统计学意义(P>0.05)。结论首次表达和鉴定了有活性的钩体胶原酶,但其在钩体病出血中的作用尚待证实和探讨。

Objective To detect in vivo and in vitro activities of Leptospira interrogans （L.interrogans） collagenase in order to identify the role of collagenase （LA0872） of Leptospira interrogans serovar Lai strain Lai in the pathogenesis of hemorrhage caused by pathogenic leptospira.Methods Recombinant la0872 was cloned and expressed in E.coli,and enzyme activity was detected by gelatin zymography.The differences in collagenase gene,transcription and enzyme activity among three leptospira strains with different virulence （L.interrogans serovar Lai strain Lai,avirulent variant strain of L.interrogans serovar Lai strain Lai and L.biflexa serovar montevalerio strain Monte Valerio） were compared.Furthermore,the changes of serum collagenase activities of guinea pigs and gerbils infected by L.interrogans serovar Lai strain Lai were detected.Results Recombinant la0872 was successfully identified with collagenase activity.There was no significant difference in collagenase gene,transcription and enzyme activity among three leptospira strains with different virulence.There was no significant difference between the serum activities of collagenase of guinea pigs and gerbils with infection by L.interrogans serovar Lai strain Lai and those without infection （P0.05）.Conclusion It is the first time to express L.interrogans collagenase and identify its activity,however,its role in leptospirosis hemorrhage still needs to be further studied.