Studies On Isolation, Identification And Biological Activity Of Rabbit Sacculus Rotundus Peptides

Stimulated the adult rabbits with bacteria ,10 hours later, killed them and take the sacculus rotundus out, triturated it and boiled for 15 minutes, mixed it with 5% acetic acid for 10 hours in 4 ℃, then centrifuged it and collected the supernate. Adjust the supernate's pH value, centrifuged it again , the supernate is the crude extract. With Sephadex G100 chromatography and collect the antibacterial components, the crude extract was purified. Tricine-SDS-PAGE analysis show that the purified sacculus rotundus peptide is only 1 protein strip. Its molecular weight is lower than 14400Da,and about 7000Da.Ultrafiltration test showed that its molecular weight is higher than 3000Da, so its molecular weight was between 3000Da to 7000Da. With Dot-ELISA assay, used antibacterial peptide antibody to detect the antibacterial peptide in purified substances. The result was positive and showed that the purified substance was antibacterial peptide.Used agarose diffusion assay and live bacteria counting assay to reveal its antibacterial activity to" 10 bacteria strains.The result showed the sacculus rutundus peptide have strong antibacterial activity to each bacteria strain.In agarose diffusion assay,7 strains appeared distinct antibacterial loop,the bacteria killing units were 0 to 104.1n live bacteria counting assay.the rate of killed bacteria of each strain was from 100% to 49.3%.This study preliminarily indicate strong antibacterial activity and wide antibacterial range of this ABP.Treated the Pseudomonas aeruginosa with ABP, use transmission electron microscope (TEM), to observe the action of ABP to bacteria. The result showed that the bacteria became smaller and thiner, the color was heavier, and vesicle-like substances appeared on the membrane of bacteria .Treated the Newcastle disease virus(NDV) and Infectious bronchitis virus(IBV) with ABP, then inoculate them into chicken embryo ,then measured the erythrocyte agglutination titer of NDV in chicken embryo allantoic fluids, and observe the pathological changes of chicken embryo. The result showed that the erythrocyte agglutination titer of NDV was lower than controlled series, and the pathological changes were less serious than controlled series, indicate that ABP can inhibit the pathological changes of chicken embryo caused by NDV and IBV.Injected the ABP to chicken, then measure the body weight; calculate the index number of thymus, spleen and bursa Fabricius; measure the intestinal villus lengh of duodenum, jejunum and ileum; measure the antibody titers of live vaccine and inactivated vaccine; count the number of intraepithelial lymphocytes of duodenum, jejunum and ileum; count the IgA positive substances of duodenum, jejunum , ileum, caecum and bursa Fabricius.to study the effect of ABP to the above target. The result showed that ABP had no significant effect to the body weight and index number of thymus and spleen; could significantly effect the index number of bursa Fabricius; could significantly inhance the lengh of intestinal villus of duodenum, jejunum ;had no significant effect to the inhancement of the lengh ofileum intestinal villus; could significantly inhance the antibody liters of live vaccine and inactivated vaccine; could significantly inhance the number of intraepithelial lymphocytes of duodenum, jejunum and ileum.At last ,the ABP could significantly inhance the number of IgA positive substances of duodenum, jejunum, caecum and bursa Fabricius ,and have no significant effect on number of IgA positive substances of ileum.On the whole, through these studies, we isolated and purified the ABP from rabbit sacculus rotundus, revealed its antibacterial and antivirus activity, and its effect on immune function of chicken. Indicated its prospect as antibacterial and antivirus drugs or as immunomodulator.