2001 Fiscal Year Final Research Report Summary

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

Research Project

Project/Area Number	12672003
Research Category	Grant-in-Aid for Scientific Research (C)
Allocation Type	Single-year Grants
Section	一般
Research Field	矯正・小児・社会系歯学
Research Institution	KYUSYU UNIVERSITY
Principal Investigator	SHIBATA Yukie Kyushu University, Faculty of Dental Science, Research Associate, 大学院・歯学研究院, 助手 (30274476)
Project Period (FY)	2000 – 2001
Keywords	Porphyromonas gingivalis / periodontal disease / bacteriophage / antisense-RNA / autolysin / microbial identification test
Research Abstract	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.