Objective: The purpose of the current study was to identify the differential genes in Porphyromonas gingivalis(Pg) virulent strain W83 and avirulent strain ATCC 33277. Methods: Using suppression subtractive hybridization (SSH) to compare Pg virulent strain W83 (tester) and avirulent strain ATCC 33277 (driver). The chromosomal DNAs were purified from Pg W83 and Pg ATCC 33277, and digested by restriction enzyme RsaI. The tester DNA samples were separated and ligated with Adaptor1 and Adaptor 2R. Two subtractive hybridization and PCR profile were performed. Tester-specific DNAs also were selectively amplified. The mixture of subtracted DNA fragments were ligated with pMD-18T Vector and transformed to competent cells of E.coli JM109. Furthermore the differential subtraction library was established. Results: 194 colonies were obtained; the positive clones were identified by PCR, sequenced, and searched homologically. Subtractive library which has high subtractive efficiency was successfully set up and 36 positive clones were screened by SSH. The fragments from 88bp to 372bp were enriched in Pg virulent strain W83 sequences which were absent from Pg ATCC 33277. Dot blot analysis confirmed that all these fragments were present in Pg W83 but absent from Pg ATCC 33277. The GenBank homology search indicated that among them, two fragments PG0043 and PG0108 were associated with evasion of Pg W83, PG0540 is related to antibiotic resistance and the products of PG0724, PG1055, and PG1701 were virulence and acquisition of peptides. PG0838, PG0839 were involved with a paralogous region. Conclusions: SSH is an effective, sensitive method to identify genetic differences between closely related bacterial strains or strains associated with virulence or genomic mutation. Comparing Porphyromonas gingivalis genomic differences and isolating pathogenicity related genes, SSH may provide an important clue for studying the mechanism of occurrence and development of periodontal disease. Supported by the National Natural Science Foundation of China (No. 30371542)
Division: IADR General Session
Meeting:2006 IADR General Session (Brisbane, Australia) Location: Brisbane, Australia
Year: 2006 Final Presentation ID:362 Abstract Category|Abstract Category(s):Periodontal Research - Pathogenesis
Authors
Lin, Li
( China Medical University, Shenyang, N/A, China
)
Pan, Ya-ping
( China Medical University, Shenyang, N/A, China
)
Li, Chen
( China Medical University, Shenyang, N/A, China
)
Li, Yanjun
( China Medical University, Shenyang, N/A, China
)