Preparation Of Egg Yolk Antibody Against Porphyromonas Gingivalis By Oleic Acid Method And Its Bactericidal Effect

ObjectivePeriodontitis is the main cause of tooth loss in adults in China.Among them,P.gingivalis is considered to be the main pathogenic bacteria causing periodontitis.Egg yolk antibodies are also known as egg yolk immunoglobulin(Immunoglobulin of yolk,Ig Y).Ig Y has strong heat resistance,acid resistance,and resistance to enzyme degradation.The preparation method is simple and can be stored at room temperature for 3 months.At the same time,Ig Y as an antibody can inhibit bacterial adhesion,bacterial enzyme activity and neutralizing toxins,and studies have shown that anti-Porphyromonas gingivalis chicken egg yolk antibody(P.gingivalis-Ig Y,P.g-Ig Y)can inhibit oral P.gingivalis proliferation.In this study,oleic acid(OA)was used for the first time to prepare anti-P.g-Ig Y,and the activity of anti-P.g-Ig Y prepared by OA method and the antibacterial function in vitro were provided,which provided an experimental basis for the future detection and therapeutic application of P.gingivalis.Methods1.Immunization of P.gingivalis.BHI culture medium was used to recover P.gingivalis ATCC 33277 and virulence strain W83.The colony liquid was enriched with 48 h of bacteria,and the strain was detected by gram staining and 16s RNA gene sequence.The identified bacteria were used for immunization.Inactivate P.gingivalis with a 10 g/L formaldehyde solution,adjust the bacterial concentration to 1×10~9 CFU/m L as the antigen.Laying hens were immunized 4 times,with 10d intervals between each other;2.Purification of Ig Y and detection of antibody activity.Ig Y was purified by the classic method-two-step ammonium sulfate precipitation method(control group)and OA method(experiment group),and the direct agglutination test was used to detect the antigen-antibody agglutination activity of Ig Y after purification by different methods;3.Purification of Ig Y activity by OA method.Indirect ELISA method was used to detect the change of antibody titer of Ig Y after OA method purification at different time points.SDS-PAGE electrophoresis and Western Blot were used to detect the molecular weight and antibody specificity of Ig Y(40 d)after OA method purification;4.Purification of anti-P.g-Ig Y on P.gingivalis biofilm by OA method.Detect the effect of anti-P.g-Ig Y(OA method,40 d)concentration on the formation of P.gingivalis biofilm by crystal violet staining;5.The effect of anti-P.g-Ig Y purified by OA method on P.gingivalis invasion of HPDLF cells.Infiltrate HPDLF cells with P.gingivalis(MOI=50:1),and add specific concentrations of anti-P.g-Ig Y(OA method,40 d)for 12 h.Observation under the microscope,bacterial staining and plate colony counting were used to detect the effects of anti-P.g-Ig Y on P.gingivalis invasion of HPDLF cells.Results1.Purification of Ig Y by OA method,21 m L of egg yolk can obtain about 190mg of total Ig Y,which is higher than the AS method,the experimental steps are simple and time-saving.The Ig Y purified by both methods specifically binds P.gingivalis;2.The titer of Ig Y purified by the OA method was as high as 1:10000,and the titer of the non-immunized Ig Y was close to 0.The subsequent experiments all used the 40 d Ig Y purified by the OA method.SDS-PAGE electrophoresis and Western Blot.The protein size was 180 k Da,mainly containing two bands of 65k Da and 27 k Da.By Western Blot detection,Ig Y specifically recognizes P.gingivalis,but has no specificity to the virulent strains W83 and ATCC 33277,consistent with direct agglutination test results;3.The results of crystal violet staining showed that compared with the blank control group,anti-P.g-Ig Y significantly inhibited P.gingivalis proliferation and biofilm formation(P<0.05).And the higher the concentration of anti-P.g-Ig Y,the more obvious the inhibitory effect of P.gingivalis on biofilm formation;4.Cell culture results showed that compared with the blank control group,the experimental group had visible agglutination in the culture medium.By staining and plate colony counting,compared with the blank control group,the experimental group had P.gingivalis invading cells(P<0.05).With the increase of anti-P.g-Ig Y concentration,the number of P.gingivalis bacteria invading HPDLF cells gradually decreased.ConclusionCompared with AS method(the classic method),the OA method can simplify the anti-P.g-Ig Y purification step,shorten the purification time,and significantly increase the yield;The anti-P.g-Ig Y purified by OA method can inhibit the proliferation and biofilm formation of P.gingivalis,and inhibit the invasion of HPDLF cells by P.gingivalis.