巴氏杆菌糖酵解酶的黏附作用研究

The adhesion activity of Pasteurella glycolytic enzymes

摘要:

巴氏杆菌（主要是多杀性巴氏杆菌）可以引起多种动物疫病（巴氏杆菌病），同时也引起人类感染发病。[目的] 研究巴氏杆菌糖酵解酶对宿主细胞（兔肾细胞）和两种常见分子[纤连蛋白（fibronectin，Fn）和血浆纤维蛋白溶解酶原（plasminogen，Plg）]的黏附作用。[方法] 采用原核表达系统对多杀性巴氏杆菌的糖酵解酶进行表达并纯化及制备多克隆抗体，通过菌体表面蛋白定位检测、黏附与黏附抑制等实验探究巴氏杆菌糖酵解酶的黏附作用。[结果] 菌体表面蛋白检测结果显示除烯醇化酶和丙酮酸激酶外的7个糖酵解酶在多杀性巴氏杆菌表面存在。这7个糖酵解酶均能黏附兔肾细胞，但仅有磷酸葡萄糖异构酶的多克隆抗体能对多杀性巴氏杆菌黏附宿主细胞产生抑制作用。Far Western blotting结果显示9个糖酵解酶均能结合宿主Fn和Plg。招募抑制实验结果显示磷酸葡萄糖异构酶、醛缩酶、磷酸甘油酸变位酶的抗体对多杀性巴氏杆菌结合Fn和Plg都有抑制作用，磷酸果糖激酶、丙糖磷酸异构酶、甘油醛-3-磷酸脱氢酶、磷酸甘油激酶抗体仅对多杀性巴氏杆菌结合Fn或Plg有抑制作用。[结论] 多杀性巴氏杆菌糖酵解酶成员葡萄糖异构酶、磷酸果糖激酶、醛缩酶、丙糖磷酸异构酶、甘油醛-3-磷酸脱氢酶、磷酸甘油激酶、磷酸甘油酸变位酶在多杀性巴氏杆菌黏附宿主细胞或分子过程中发挥作用。该研究的完成将加深巴氏杆菌病分子发病机制的认识，并为巴氏杆菌病的诊断标识筛选、新型疫苗创制和药物靶标筛选等提供基础数据。

关键词:巴氏杆菌;糖酵解酶;黏附;纤维连接蛋白;血浆纤维溶解酶原

Abstract:

Pasteurella, mainly Pasteurella multocida, can cause a variety of diseases (pasteurellosis) and also human infection and diseases.[Objective] The aim of this work was to characterize the adhesion activity of Pasteurella glycolytic enzymes to host cells (rabbit kidney cells) and two common host molecules, plasminogen (Plg) and fibronectin (Fn).[Methods] The glycolytic enzymes of P. multocida were expressed by prokaryotic expression system, then purified, and their polyclonal antibodies were prepared. The adhesion activity of P. asteurella glycolytic enzymes was studied by cell surface localization detection, adhesion and adhesion inhibition experiments.[Results] Seven glycolytic enzymes except enolase and pyruvate kinase were found on the surface of P. multocida cells. The seven glycolytic enzymes all could adhere to rabbit kidney cells, but only PGI polyclonal antibodies could inhibit the adhesion of P. multocida. Far Western blotting showed that all nine glycolytic enzymes could bind to the host Plg and Fn. The recruitment inhibition experiments showed that the antibodies of phosophoglucose isomerase, aldolase and phosphoglycerate mutase could inhibit the binding of P. multocida to both Plg and Fn, while the antibodies of phosphofructokinase, triose phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase could only inhibit the binding of P. multocida to either Plg or Fn.[Conclusion]] The phosophoglucose isomerase, phosphofructokinase, aldolase, triose phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase and phosphoglycerate mutase of P. multocida play an important role in the adhesion of P. multocida to host cells or molecules. The completion of this study will deepen the understanding of molecular pathogenesis of Pasteurellosis, and provide basic data for new diagnostic markers, new vaccines, and new drug targets.

Key words:Pasteurella multocida;glycolytic enzyme;adhesion;fibronectin;plasminogen