Molecular biological research for Periodontal pathogenic surface antigens of Campylobacter rectus.

直肠弯曲杆菌牙周病原表面抗原的分子生物学研究。

• 批准号: 13671913
• 负责人: MIURA Tadashi
• 金额: $ 1.6万
• 依托单位: Tokyo Dental College
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13671913/
• 关键词: Campylobacter rectus; monoclonal antibody; Infection; Bacteroides forsythus; cloning; Periodontal bacteria; Periodontal lesion; detection; Bacteroides forsythus; C.rectus; monoclonal antibody; cloning; 嫌気性細菌

In the diagnosis of periodontal disease, microbial examinations can identify patients and periodontal sites that are undergoing active tissue destruction and can aid in understanding of fluctuations of periodontopathogens after treatment. Porphyromonas gingivalis, Actinobacillus actinornycetenicomitans, Fusobacterium nucleatum Bacteroides forsythus, Carnpylobacter rectus are known as major periodontal bacteria. Campylobacterrectushas often been detected in large numbers in deeper subgingival pockets and has been implicated in adult periodontitis and rapidly advancing periodontitis. It is gram-negative, microaerophilic bacterium, a few reports for clinical status and C.rectus were submitted. Therefore, the aim of this study is to clarify its pathogens.1) We cultured successfully a great deal of Carnpylobacter rectus cells as a result of reforming a broth.2) We prepared monoclonal antibodies and polyclonal ones. Campylobacter rectus ATCC 33238 whole cells were used as the immunogen.3) We isolated three monoclonal antibodies specific to C.rectus. These antibodies recognized a peculiar 150 KDa protein of C.rectus, thought a S-layer. One of the antibodies did not cross-react to some clinical strains, showed that the S-layer has heterogeneity.4) As a result that relationship between PD and the detection rate of C.rectus using dot-blot analysis was statistically significant. As periodontal pockets became deeper, the detection rate of C.rectus increased.5) With one of the polyclonal antibodies, we made an attempt of cloning C.rectus genes from chromosomal DNA fragments treated by restriction enzyme. As a result, we got positive clones. We determined its DNA sequences of one of the clones. This gene had a specific sequence, and not homologous to known genes.6) Furthermore in order to clarify C.rectus pathogenecities, we are planning to continue gene-cloning, and simultaneously we analyse Bacteroides forsythus cell surface antigens using several monoclonal antibodies.

在牙周病的诊断中，微生物检查可以识别患者和正在经历活跃组织破坏的牙周部位，并有助于了解治疗后牙周病原体的波动。牙龈卟啉单胞菌（Porphyromonas gingivalis）、放线菌（Actinobacillus actinornycetenicomitans）、具核梭杆菌（Fusobacterium nucleatum）、类杆菌（Bacteroides）、直肉杆菌（Carnpylobacter rectus）是已知的主要牙周细菌。弯曲杆菌经常在较深的龈下袋中大量检出，并与成人牙周炎和快速进展的牙周炎有关。它是革兰氏阴性，微需氧菌，提交了一些关于临床状况和直肌梭菌的报告。因此，本研究的目的是阐明其致病机理。1）通过改良培养液，成功培养了大量的直肌肉杆菌细胞。以直弯杆菌ATCC 33238全细胞为免疫原。3）分离出三株直弯杆菌特异性单克隆抗体。这些抗体识别一种特殊的150 KDa的蛋白质，认为是一个S层。其中1株抗体与部分临床菌株无交叉反应，表明S层存在异质性。4）斑点杂交分析结果表明PD与直链念珠菌检出率之间存在统计学意义。随着牙周袋深度的增加，直肌隐孢子虫的检出率也随之增加。5）利用其中一株多克隆抗体，尝试从限制性内切酶处理的染色体DNA片段中克隆直肌隐孢子虫基因。结果，我们得到了阳性克隆。我们确定了其中一个克隆的DNA序列。该基因具有特定的序列，与已知基因不同源。6）此外，为了阐明直链梭菌的致病性，我们计划继续进行基因克隆，同时使用几种单克隆抗体分析Bacteroides rectus细胞表面抗原。

项目成果

H.Ihara: "Detection of Campylobacter rectus in periodontitis sites by monoclonal antibodies"Journal of Periodontal Research. (2001)

H.Ihara：“通过单克隆抗体检测牙周炎部位的直肠弯曲杆菌”牙周研究杂志。

H.Ihara: "Detection of Campylobacter rectus in periodontitis sites by monoclonal antibodies"Journal of Periodontal Research. 38. 64-72 (2003)

H.Ihara：“通过单克隆抗体检测牙周炎部位的直肠弯曲杆菌”牙周研究杂志。