Characterization of Heat Shock Proteins from Periodontopathogenic Bacteria and Their Virulences

牙周病原菌热激蛋白的表征及其毒力

基本信息

批准号：
08672372
负责人：
HINODE Daisuke
金额：
$ 1.47万
依托单位：
The University of Tokushima
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

项目摘要

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

最近，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相当多的关注。在这项研究中，我们建立了从牙周病原细菌中分离热休克蛋白的方法，并使用特异性抗体研究了它们的免疫学特征。 .通过亲和层析从牙龈卟啉单胞菌、伴放线放线杆菌和福赛斯拟杆菌的全细胞中分离热休克蛋白。腺苷5'-三磷酸琼脂糖，然后进行制备型聚丙烯酰胺凝胶电泳，可以得到大量纯化的GroEL-和DnaK-样蛋白，并且这些蛋白可以成功地用于获得具有高滴度抗体和非常好的兔血清。通过Western-immunoblotting，发现P.gingivalis的GroEL样蛋白定位于细胞质，DnaK样蛋白定位于细胞质。 f … 更多 rom P.gingivalis 定位于细胞质和周质。对 P.gingivalis 和 B.forsythus 的每个 DnaK 样蛋白的 N 端氨基酸序列分析表明，其与埃希氏菌的 DnaK 蛋白具有高度同源性然而，通过斑点印迹法，这两种 HSP 与商业抗 DnaK 多克隆抗体的反应非常弱。分离出的 GroEL 样蛋白P.gingivalis、A.actinomycetemcomitans 和 B.forsythus 显示出它们的 N 末端氨基酸序列的高度同源性。针对每种 GroEL 样蛋白产生的多克隆抗体显示出这些抗体针对重组体的高水平交叉反应性。人类 HSP60 较弱。我们的研究结果表明，来自牙周病原菌的 DnaK 和 GroEL 样蛋白非常保守，并且 GroEL 样蛋白彼此相似更仔细地说，可能表明来自细菌的热休克蛋白是免疫反应的重要目标，然而，这些抗感染免疫反应可能会通过其分子模拟从自身保护转向发病机制。