Egg yolk-derived immunoglobulin (IgY) against Porphyromonas gingivalis 40-kDa outer membrane protein inhibits coaggregation activity.

The anaerobic bacterium Porphyromonas gingivalis, a major pathogen in periodontitis, aggregates with a number of oral bacteria to form dental plaque, which is important for its colonization. We previously cloned the gene coding the 40-kDa outer membrane protein (OMP) of P. gingivalis 381 and produced large amounts of the recombinant (r) protein. Affinity-purified rabbit antiserum against r40-kDa OMP effectively inhibited the coaggregation activity of P. gingivalis to oral bacteria, thus 40-kDa OMP was thought to be an important coaggregation factor of P. gingivalis. Further, since it is conserved among many P. gingivalis strains, this coaggregation factor may be an effective target for passive immunotherapy against P. gingivalis infection. Recently, passive immunization approaches using a specific antibody produced from hen egg yolk (IgY) have been developed for oral infectious diseases, and shown to be convenient and economic. In the present study, we immunized hens intramuscularly with r40-kDa OMP and obtained highly purified IgY from the egg yolks. The purified IgY specifically recognized r40-kDa OMP and also reacted with a functional coaggregation-associated domain peptide of 40-kDa OMP. Our results demonstrated that a ratio of purified IgY as low as 2.5 microg/150 microl significantly inhibited the coaggregation of P. gingivalis with Streptococcus gordonii, which was verified by a visual coaggregation assay and radioactivity-based quantitative micro-coaggregation assay. We concluded anti-r40-kDa OMP IgY may be useful for passive immunization against periodontal diseases caused by P. gingivalis infection.

Inhibition of Porphyromonas gingivalis hemagglutinating activity by IgY against a truncated HagA.

Porphyromonas gingivalis has been implicated as an important pathogen in the development of periodontitis. Hemagglutinins have been identified as important adhesion molecules, allowing Porphyromonas gingivalis to adhere to gingival tissue cells, and to attach and lyse erythrocytes in order to uptake Fe ions as essential nutrition. One hemagglutinin, hemagglutinin A (HagA), has been molecularly cloned via functional screening for hemagglutinating activity. We previously cloned the gene encoding the 200-kDa cell-surface antigenic protein that was reacted by sera from periodontitis patients and was identified as a truncated protein of HagA by nucleotide sequence analysis. We further subcloned the gene encoding an 122-kDa protein (122k-HagA) which is a fusion protein composed of an 80-kDa truncated HagA containing the functional motif PVQNLT and a 42-kDa maltose binding protein. Passive immunization against infectious pathogens by specific antibodies produced from hen egg yolk antibody (IgY) has been extensively developed. In the present study, to develop passive immunotherapy against periodontal disease, we purified the recombinant 122k-HagA and used this to immunize hens and produce IgY. The purified IgY reacted with the recombinant 122k-HagA and the synthetic peptide containing PVQNLT, and inhibited hemagglutinating activity of Porphyromonas gingivalis. Thus, the novel IgY may be useful in the development of a passive immunization against periodontal diseases caused by P. gingivalis infection.

Further development of an electroosmotic medium pump system for preparative disk gel electrophoresis.

A simple and practical 6.8-cm-diameter (36.30-cm(2) cross-sectional-area) preparative disk gel electrophoresis device, based on the design of M. Hayakawa et al. (Anal. Biochem. 288 (2001) 168), in which the elution buffer is driven by an electroosmotic buffer flow through the membrane into the elution chamber from the anode chamber was constructed. We have found that the dialysis membranes employed provide suitable flow rates for the elution buffer, similar to those of an earlier 3.6-cm-diameter device, resulting in the prevention of excess eluate dilution. The efficiency of this device was demonstrated by the fractionation of a bovine serum albumin (BSA) Cohn V fraction into monomer, dimer, and oligomer components using nondenaturing polyacrylamide gel electrophoresis (native-PAGE). The maximum protein concentration of the eluate achieved was 133 mg/ml of BSA monomer, which required a dilution of the eluate for subsequent analytical PAGE performance. As a practical example, the two-dimensional fractionation of soluble dipeptidyl peptidase IV (sDPP IV) from 50 ml fetal bovine serum (3.20 g protein) per gel is presented. The sDPP IV enzyme protein was recovered in a relatively short time, utilizing a 6.5% T native-PAGE and subsequential sodium dodecyl sulfate-PAGE system. This device enhances the possibility of continuous electrophoretic fractionation of complex protein mixtures on a preparative scale.