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【目的】肺炎克雷伯菌(K.pn)与宿主细胞的粘附是致病的首要条件,粘附过程主要通过菌毛粘附素MrkD蛋白介导。为了进一步分析MrkD蛋白与宿主细胞间的粘附机制,进一步确定MrkD蛋白的粘附阻断作用。【方法】构建肺炎克雷伯菌菌毛粘附素融合蛋白原核表达质粒pGEX-4T-mrkD,转入大肠杆菌BL21,优化诱导表达条件,表达产物经亲和层析纯化、凝血酶切除融合蛋白GST标签后,进行SDS-PAGE和Westernblot鉴定。激光共聚焦显微镜定位MrkD蛋白在宿主细胞上的结合部位;通过粘附活性试验与粘附动力学试验研究了MrkD蛋白的生物活性。【结果】试验得到了分子量为35kDa的MrkD蛋白,定位了MrkD蛋白在宿主细胞上的结合部位,并证明了MrkD蛋白可以显著影响肺炎克雷伯菌对宿主细胞的粘附力。【结论】本试验首次证实了MrkD蛋白的粘附阻断作用并观察到其与宿主细胞的作用位点,为研究肺炎克雷伯菌的致病机制,寻找粘附素功能表位奠定了基础。
【Objective】 The adhesion of K. pneumoniae to host cells is the primary condition for pathogenicity. The adhesion process is mainly mediated by the piliated adhesin MrkD protein. In order to further analyze the adhesion mechanism between MrkD protein and host cells, we further determined the blocking effect of MrkD protein. 【Method】 The prokaryotic expression plasmid pGEX-4T-mrkD was constructed and transfected into E. coli BL21 to optimize the expression conditions. The expressed product was purified by affinity chromatography and cloned by thrombin Protein GST tag, the SDS-PAGE and Westernblot identification. Laser confocal microscopy was used to locate the binding site of MrkD protein on host cells. The biological activity of MrkD protein was studied by adhesion activity assay and adhesion kinetics assay. 【Result】 The results showed that MrkD protein with a molecular weight of 35 kDa could locate the binding site of MrkD protein on host cells and proved that MrkD protein could significantly affect the adhesion of Klebsiella pneumoniae to host cells. 【Conclusion】 This experiment confirmed for the first time that MrkD protein blocks the adhesion and observes its interaction with host cells. This study laid the foundation for studying the pathogenesis of Klebsiella pneumoniae and finding the functional epitope of adhesin .