Counteractive therapy against periodontal bacteria by introduction of antisense-RNA of virulent genes

通过引入毒力基因的反义RNA对抗牙周细菌的治疗

• 批准号: 12672003
• 负责人: SHIBATA Yukie
• 金额: $ 2.24万
• 依托单位: KYUSYU UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12672003/
• 关键词: Porphyromonas gingivalis; periodontal disease; bacteriophage; antisense-RNA; autolysin; microbial identification test; プロモーター; CATアッセイ

First, transcriptional levels of the Porphyromonas gingivalis pathogenic genes that had been cloned previously were compared by CAT assay. There was no significant difference among them. Next, though the bacteriophage infectable to P. gingivalis was tried to be isolated from the culture by ultraviolet and mitomicin C treatments, the attempt was failed. Isolation of autolysins of P. gingivalis as a new target was tried by the plate assay method. P. gingivalis was spread on enriched BHI agar containing P. gingivalis cells (final OD_<550>; 2.0) and incubated at 37℃ for 18 h. No clear zone was formed around a P. gingivalis colony. The gene homologous to the bacteriophage gene was searched for the P. gingivalis chromosomasl DNA database and the ampD gene that encodes N-acetylmuramoyl-L-alanine amidase was found. This gene was amplified by polymerase chain reaction (PCR) and inserted into pBluescriptII KS+. The resultant plasmid (pPGAMP) was introduced into Escherichia coli K12. This transformant was spread on enriched BHI agar containing P. gingivalis cells. A small clear zone was formed around a transformant colony. Experiments to increase the transcriptional level of the ampD gene are currently in progress. Also, the microbial identification test was performed on saliva and gingival crevicular fluid of 60 patients by using PCR method. Saliva was found to be an effective sample for examination of occurrence of P. gingivalis as well as gingival crevicular fluid. The relationship between bleeding on probing and P. gingivalis was significant as well as the relationship between pocket depth and P. gingivalis.

首先，通过CAT测定比较先前克隆的牙龈卟啉单胞菌致病基因的转录水平。他们之间没有显着差异。接下来，虽然尝试通过紫外线和丝裂霉素C处理从培养物中分离可感染牙龈卟啉单胞菌的噬菌体，但该尝试失败了。通过平板测定法尝试分离牙龈卟啉单胞菌自溶素作为新靶标。将牙龈卟啉单胞菌涂布在含有牙龈卟啉单胞菌细胞（最终OD_<550>；2.0）的富集BHI琼脂上，并在37℃下孵育18小时。牙龈卟啉单胞菌菌落周围没有形成清晰的区域。在牙龈卟啉单胞菌染色体DNA数据库中检索与噬菌体基因同源的基因，发现编码N-乙酰胞壁酰-L-丙氨酸酰胺酶的ampD基因。该基因通过聚合酶链式反应 (PCR) 扩增并插入 pBluescriptII KS+ 中。将所得质粒(pPGAMP)导入大肠杆菌K12。将该转化体铺展在含有牙龈卟啉单胞菌细胞的富集BHI琼脂上。在转化体菌落周围形成小的透明区域。目前正在进行提高 ampD 基因转录水平的实验。采用PCR方法对60例患者的唾液和龈沟液进行微生物鉴定检测。唾液被发现是检查牙龈卟啉单胞菌以及龈沟液的有效样本。探诊出血与牙龈卟啉单胞菌之间的关系以及袋深度与牙龈卟啉单胞菌之间的关系均显着。