Functional Domain of Hemagglutinating Activity in P. gingivalis 40kDa-OMP

Objectives: 40kDa outer membrane protein (OMP) has been molecular cloned from P. gingivalis 381. Since the antibody against recombinant 40kDa-OMP inhibited the hemagglutinating activity and the polymeric r40-kDa-OMP expressed hemagglutinating activity, the 40kDa -OMP was thought to be one of hemagglutinins. To develop passive immunotherapy against periodontitis, MAbs were constructed by immunization with r40kDa-outer membrane protein and examined those inhibitory effect on hemagglutinating activity. Furthermore, functional domain expressing hemagglutinating activity was identified. Methods: Highly purified r40kDa-OMP was injected intraperitoneally into BALB/c mice, spleen derived cells were collected and combined with P3U1 myeloma cells. After selection of hybridoma cells using HAT medium, clones producing neutralizing hemagglutination activity were isolated by a single manipulation. A phage-displayed epitope mapping system was used to examine the functional domain expressing hemagglutinating activity by biopanning using the neutralizing MAb. Peptides mapped the relevant molecular within a short stretch corresponding to 40-kDa-OMP amino acid sequence was chemically synthesized and determine its inhibitory effect on hemagglutinating activity. Results: MAbs against r40-kDa-OMP were capable inhibiting hemagglutinating activity. Synthetic peptides, corresponding to residues WPRVGQLFIALDQTLGIPTFSVCRME in 40kDa-OMP, abolished hemagglutinating activity in a dose-dependent manner. Conclusions: MAbs and the functional domain drived synthetic peptide may be useful to preventing periodontal diseases caused by P. gingivalis infection.