Characterization of Heat Shock Proteins from Periodontopathogenic Bacteria and Their Virulences

近年来，热休克蛋白（HSP）在炎症和自身免疫性疾病中的潜在作用受到了广泛关注。本研究建立了牙周致病菌热休克蛋白（HSP）的分离方法，并利用特异性抗体对其免疫学特性进行了研究，从牙龈卟啉单胞菌（Porphyromonas gingivalis）全细胞中分离HSP，放线共生放线杆菌和拟杆菌的亲和层析5 '-三磷酸腺苷，琼脂糖凝胶电泳，可获得大量纯化的GroEL和DnaK样蛋白。Western免疫印迹结果显示，牙龈卟啉单胞菌的GroEL样蛋白定位于胞浆，DnaK样蛋白定位于胞 ...更多信息 牙龈卟啉单胞菌和B. coli的DnaK蛋白N-末端氨基酸序列分析表明，它们与大肠杆菌的DnaK蛋白有很高的同源性，但这两种热休克蛋白与抗DnaK多克隆抗体的反应很弱。伴放线菌与B. patienthus的N-末端氨基酸序列具有高度的同源性。针对每个GroEL样蛋白的多克隆抗体显示出高水平的交叉反应性。这些抗体对重组人HSP 60的反应性很弱。我们的研究结果表明，DnaK和GroEL样蛋白从牙周病病原体是很好的保守和GroEL样蛋白彼此相似更密切。这可能表明来自细菌的热休克蛋白是免疫应答的重要靶点，然而，这些抗感染免疫应答可能通过其分子模拟从其自身的保护转变为致病。少

Recently, considerable attention has been given to the potential roles of heat shock protein (HSP) of in inflammation and autoimmune diseases. In this study, we established the isolation procedure of HSP from periodontopathogenic bacteria and investigated the immunological characterization of them using the specific antibodies.The isolation procedure of HSPs from whole cells of Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans and Bacteroides forsythus was performed by affinity chromatography on adenosine 5'-triphosphate-agarose followed by preparative polyacrylmide gel electrophoresis and could obtain the considerable amount of purified GroEL- and DnaK-like proteins. Also, these proteins could be successfully used to obtain rabbit sera which possessed high titer of antibodies and a very good specificities comparing to the commercial ones.By Western-immunoblotting, it found that the GroEL-like protein from P.gingivalis was localized in cytoplasm and the DnaK-like protein f … More rom P.gingivalis was localized in both cytoplasm and periplasm.Analysis of the N-terminal amino acid sequence of each DnaK-like protein from P.gingivalis and B.forsythus showed a high degree of homology with the DnaK protein from Escherichia coli.However, these two HSPs reacted very weakly with a commercial anti-DnaK polyclonal antibody by dot-blotting.GroEL-like proteins isolated P.gingivalis, A.actinomycetemcomitans and B.forsythus showed a high degree of homology of their N-terminal amino acid sequences. Polyclonal antibodies raised against each GroEL-like protein showed a high level of cross-reactivity. Reactivity of these antibodies against recombinant human HSP60 was weak.Our findings suggest that DnaK- and GroEL-like proteins from periodontopathogens are well conserved and that the GroEL-like protein resemble each other more closely. It is possible to suggest that HSPs from bacteria are important targets of the immune response, however, these anti-infectious immune response may shift from their own protection to pathogenesis by their molecular mimicry. Less