PUTATIVE DOWN-STREAM SIGNALING MOLECULE OF GTPase IN Porphyromonas gingivalis

摘要

Porphyromonas gingivalis is a strict anaerobic bacterium mainly responsible for periodontal disease in oral cavity. Putative GTPase gene (pgp) of this bacterium was cloned and its recombinant protein (rPG P) was produced in Escherichia coli. Based onthe amino acid sequence of SGP that is a GTP-binding protein of Streptococcus mutans, putative GTPase amino acid sequence was deduced in the data base of genome sequences of Porphyromonas gingivalis. A 900-bp PGR fragment was amplified with P. gingivalisgenomic DNA as a template and cloned into E. coli J M109. Then pgp was transferred into pQE-30 expression vector to make pQE-PGP for production of rPGR. This protein was produced and purified by Ni-NTA affinity column chromatography. Anti-PG P antibodywas also produced in Sprague Dawley rats. Using Westernblot analysis with this antibody, it was confirmed that the rPGP produced in E. coli was identical to that of donor strain. Furthermore, by Southernblot analysis it was revealed that the pgp was originated from P. gingivalis. By immunopre-cipitation with anti-PGP antibody and N-terminal amino acid sequence analysis it was found that PGP was able to bind to acetate kinase, which was reported to be a secondary signaling-molecule in anaerobic microorganisms. Therefore, these results imply that P. gingivalis produces putative GTPase and this protein might play a potential role in signaling pathway in oral biofilm formation.