肺炎克雷伯菌菌毛粘附素克隆表达及其对体外培养细胞的粘附活性

摘要：【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Western blot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实验研究了MrkD蛋白的生物活性。【结果】实验得到了分子量为35 kDa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了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。

MrkD adhesin of Klebsiella pneumoniae expression, purification and analysis of adhesive activity

Abstract: [Objective] The mrkD gene encodes the adhesin which mediates Klebsiella pneumoniae to adhere human respiratory tissue.We aimed to analyze the adhesion mechanism and adhesion block funcntion of MrkD adhesin. [Methods] The recombinant glutathione-S-transferase(GST)-tagged adhesive protein(MrkD) was expressed in E. coli and was purified to homogeneity using GST affinity chromatography. The GST tag was cut by thrombin to obtain the MrkD protein that was identified by SDS-PAGE and Western blot. The adhesive activity of MrkD was examined by adhesive experiments and the binding site was observed by laser confocal microscopy. [Results] The adherent activity of Klebsiella pneumoniae was significantly inhibited by the MrkD. These experimental data demonstrated that the MrkD inhibited the adhesion of Klebsiella pneumoniae. [Conclusion] Our results suggest that MrkD adhesin contains the adhesion epitopes.The future work will be carried out to identify the epitopes and characterize them, then to optimize the combination presentation of these epitopes to develop an efficient vaccine for Klebsiella pneumoniae.