Analysis of major outer membrane proteins from periodontopathogen and relationship between these proteins and clinical symptom in periodontitis

1.不同牙龈假单胞菌菌株主要外膜蛋白的分析。主要外膜蛋白，如RagA和Pgm6/7，在所有菌株中均以相似的分布模式被发现。但不同菌株间RagB的移动性和对特异性抗体的反应不同，表明RagB具有异质性。培养环境因素对牙龈假单胞菌主要外膜蛋白表达的影响研究了几种因素对牙龈假单胞菌主要外膜蛋白表达的影响，结果表明温度是影响牙龈假单胞菌主要外膜蛋白表达的最主要因素。随着温度的升高，RagA、RagB、龈痛以及毛、短毛的表达均减少。然后，我们采用连续培养，使培养环境的调节成为可能。随着生长速度的增加，毛和短毛的产量增加，而牙龈蛋白酶的数量和活性降低。在营养贫乏的培养基中，被毛和短被毛减少。当环境由无氧转变为好氧时，牙龈炎的活性降低。通过二维凝胶电泳观察氧化应激下蛋白质数量增加的斑点，并通过质谱鉴定。利用基因缺失突变体表征主要外膜蛋白。外膜形成异源三聚体中存在与大肠杆菌OmpA蛋白同源的主要外膜蛋白Pgm6/7。由于电镜观察到Pgm6/7基因缺失突变体中有许多囊泡样结构，Pgm6/7可能参与了外膜完整性的稳定。由于RagA和ragb缺失突变体的构建已经完成，我们正在对突变体进行表征。牙周炎患者血清对牙龈假单胞菌主要外膜蛋白的免疫反应。为了检测牙龈假单胞菌的免疫反应性抗原，我们使用了来自根尖周炎患者的血清。半数以上血清对牙龈假单胞菌细胞组分，尤其是外膜蛋白RagB和牙龈蛋白酶有较强的免疫反应。临床症状如打击声、根尖周围半透明等与对牙龈卟啉菌的免疫反应有关。少

1.Analysis of major outer membrane proteins among various P.gingivalis strains.Major outer membrane proteins, such as RagA and Pgm6/7,were found in all strains tested in a similar distribution pattern. However, mobility and reaction of RagB against specific antibody was different among strains, indicating RagB heterogeneity.2.Effect of culture environment factors on the expression of P.gingivalis major outer membrane proteinsWhen effect of several factors was examined on the expression of major outer membrane proteins, temperature was the most important factor. As temperature increased, expression of RagA,RagB and gingipains as well as fimbriae and short fimbriae decreased.Then, we used continuous culture in which regulation of culture environment was possible. With increase of growth rate, production of fimbriae and short fimbriae increased, while amount and activity of gingipains decreased. In a nutrient-poor medium, fimbriae and short fimbriae decreased. When atmosphere was changed … More from anaerobic to aerobic condition, activity of gingipains decreased. Protein spots whose amount increased under oxidative stress was observed by 2-dimensional gel electrophoresis, and those were identified by mass spectrometry3.Characterization of major outer membrane proteins using gene-deletion mutants.Major outer membrane proteins Pgm6/7, which were homologous to OmpA protein from Escherichia coli, existed in outer membrane forming heterotrimers. Since numerous vesicle-like structures were observed in the case of Pgm6/7 gene-deletion mutants by electron microscopy, Pgm6/7 might be involved in stabilization of outer membrane integrity. Since construction of RagA and RagB-deletion mutants have been finished, we are characterizing the mutants.4.Immunological reactions of sera from patients with periodontitis to P.gingivalis major outer membrane proteins.To examine immunoreactive antigens in P.gingivalis, sera from patients with periapical periodontitis were used. More than half of the sera showed strong immunological reaction to P.gingivalis cell components, especially, outer membrane proteins RagB and gingipains. Clinical symptoms such as percussion and periapical translucency were related to immnoreactivity to P.gingivalis. Less