【摘要】 目的通过克隆、表达问号钩端螺旋体赖型赖株的胶原酶基因colA(LA0872),以及制备抗胶原酶的多克隆抗体,观察胶原酶在钩体病豚鼠模型的组织中的表达,为研究其致病机制奠定基础。方法以问号钩端螺旋体赖型赖株基因组DNA为模板,用PCR扩增colA,产物克隆到表达载体pET-28b(+)中,构建含colA的重组质粒;将重组质粒转入E.coli Rosetta(DE3)菌株内,IPTG诱导表达后用Ni-NTA His-Bind亲和层析柱纯化重组蛋白;用纯化的重组蛋白免疫家兔制备抗胶原酶的多克隆抗体;Max Vision二步法染色检测胶原酶在感染钩端螺旋体的豚鼠肺组织中的分布。结果克隆表达目的基因,并成功制备了抗胶原酶的多克隆抗体。免疫组化显示在出血的肺组织中存在胶原酶。结论钩体病豚鼠模型的肺组织中有胶原酶的表达,并且存在于有钩端螺旋体分布的部位。本研究为进一步探讨胶原酶在钩端螺旋体侵袭和宿主组织出血中所起的作用奠定了基础。更多还原

【Abstract】 To investigate the expression of collagenase in a guinea pig model of leptospirosis,colA(LA0872) from Leptospira interrogans strain Lai was cloned and expressed in E.coli,and the rabbit anti-collagenase polyclonal antibody was prepared.The gene encoding for collagenase was amplified from genomic DNA of Leptospira interrogans strain Lai by using PCR,And the PCR product digested with BamH I and Hind Ш was cloned into the pET-28b(+) expression vector.The recombinant plasmid containing the right insertion was sequenced to confirm its identity and transformed into E.coli Rosetta(DE3).Upon IPTG induction,the recombinant pET-28b(+)-colA produced a new protein and rabbit anti-collagenase polyclonal antibody was preprared by subcutaneously immunizing rabbits with the protein purified with Ni-NTA His-Bind resin.The expression of collagenase from Leptospira interrogans was detected in the hemorrhagic lung tissue of guinea pigs infected with Leptospira interrogans strain Lai by MaxVision staining.It was demonstrated that the expression of collagenase from Leptospira interrogans was detected in the lungs of a guinea pig model of leptospirosis,where the leptospirae distributed.These results would provide a basis for in-depth studies for the contribution of collagenase to the invasion and spread of Leptospira interrogans and hemorrhage in host tissues.更多还原