Preparation And Studies On Egg Yolk Antibody Against Helicobacter Pylori

In this paper, the preparation of immunoglobulin Y against Helicobacter pylori named IgY-HP was studied, high purified IgY-HP was prepared from the egg yolk of hens immunized with compound antigen of supernatant from HP broken by ultrasonic wave and urease purified form H.pylori, then IgY-HP's bioactivity and functional property was evaluated.As a result of study of optimal cultivation for H.pylori, H.pylori was cultured in Columbia agar supplemented with sheep brood , growth conditions were 5%O2,85%N2,10%CO2. H.pylori strains were identified ,its spiral and curving shape was seen as a typical shape, but sometimes, the bacterial cells were rod-like or spHere-like. urease test showed its high urease activity and gram staining revealed it is a Gram-negative bacterium.Urease as a main antigen in supernatant from H.pylori broken by ultrasonic wave was purified by anion-exchange chromatograpHy and gel-permeation, the purity of urease obtained by this method was very high,the overall protein recovery was 3.05％ ,specific activity was 81.491 U/mg；Urease in supernatant from H.pylori broken by ultrasonic wave can also be purified by removing the major contaminants in supernatant prior to the ion exchange separation, the purity of urease obtained by this method was high,the overall protein recovery was 3.88％,specific activity was 87.351U/mg.. The purified urease showed the typical subunits of Mr 65kD and Mr 31kD by SDS-PAGE。After immunization of hen with compound antigen of supernatant from HP broken by ultrasonic wave and urease purified form H.pylori, the IgY-HP was extracted by water dilution and ultrafiltration method and purified by ammonium sulpHate precipitation and anion-exchange chromatograpHy, the extraction rate was 3.2％ , purity of the IgY-HP was 84.3％ after ammonium sulpHate precipitation and 97.3％ after nion-exchange chromatograpHy.The IgY and IgY-HP were examined for immunological properties by ELISA. result indicated that the titer for IgY-Hp was 32 times higher than that for IgY in egg yolk from nonimmunized hens and the titer for Urease was 16 times.Heat and pH stability test showed that the effect of pH on IgY-HP activity had relation with temperature, Along with the rising of the temperature, the IgY stability became more and more bad, at 37℃,The antibody activity of IgY-HP began to decrease at pH 4.0 and lost 60%～80% of its initial activity at pH3.0,the activity was almost lost when the value of pH decrease to 2.0. Imitating the gastric environment, the pepsin stability of IgY-HP was studied, result showed that the effect of pepsin on IgY-HP activity had relation with pH, antibody activity of the IgY-HP was almost lost when incubated with pepsin at pH2.0,while at pH4.0 and 6.0 respectively, pepsin had no notable effect on activity of IgY-HP.IgY-HP may be applied to healthcare of mankind and the antibacterial immune treatment for gastric disease associated with HP because of its effect of passive immunization by oral administration.