The Preparation And Study Of Anti-Porphyromonas Gingivalis IgY In Vitro

Objectives To obtain egg yolk antibody (IgY) in hen eggs laid by hens immunized with the protein of Porphyromonas gingivalis. To generate and purify IgY against Porphyromonas gingivalis(anti-Porphyromonas gingivalis IgY), identify its specificity, find out the variation regularity of the antibody titer in the eggs with immunization time, temperature and pH, evaluate the physicochemical character of IgY, and its inhibition the growth of Porphyromonas gingivalis in vitro.Methods Porphyromonas gingivalis ATCC 33277 were cultured under standard anaerobic conditions and harvested after increasing. Then Porphyromonas gingivalis were extracted by sonication until the cell pellets were shattered completely. After centrifugatiton, collected the supernatant which was the protein of Porphyromonas gingivalis. Five-month-old Roman hens kept in conventional isolated poultry housing were immunized for egg antibody production. The hens were inoculated intramuscularly and subcutaneously in the breast from multiple spot with 1.0ml of a vaccine consisting of oil-adjuvant protein which was mixed with 1ml protein of Porphyromonas gingivalis and 1ml Freund's adjuvant complete every 10 days, 4 times totally. The eggs were collected after the first immunization and stored at 4℃. Extracted and purified the anti-Porphyromonas gingivalis IgY after immuned in different times, protein concentration was tested by BCA, the specificity of IgY was analyzed by SDS-PAGE, the titre of IgY and its physicochemical character were evaluated by indirect ELISA, meanwhile, inhibiting growth of Porphyromonas gingivalis in vitro were tested in liquid anaerobic culture.Results The concentration of obtained anti-Porphyromonas gingivalis IgY was 2.05mg/ml by immuned. SDS-PAGE analysis of the anti-Porphyromonas gingivalis IgY showed that the molecular weight of IgY was as the same as expected. Anti-Porphyromonas gingivalis IgY appeared approximately 5 days after the first immunization, and reached the peak at about 50-55 days. Antibody titres can reach 1:100 000. Each egg can produce more than 10mg IgY, and its purification may be up to 95% as well. The most effective concentration of IgY inhibiting Porphyromonas gingivalis was 1.0 mg/ml to reach the peak, the suppression rates of Porphyromonas gingivalis growth was 47.21%.Conclusions Layer hens immuned by Anti-Porphyromonas gingivalis IgY may provide specific IgY of high titre and high concentration. The antibodies with good stability could inhibit the Porphyromonas gingivalis growth obviously.