Preliminary Identification Of Antimicrobial Peptides From Duck Leukocyte And Effects Of Crude Extract On Immunity Of Mice

Yingtaogu duck was selected to extract and isolate antimicrobial peptides from leukocyte and study the effects of crude extract on immunity of mice.Here are two series in this study.SeriesⅠExtraction,isolation and preliminary identification of antimicrobial peptides from duck leukocyteThe fresh red blood cells of Yingtaogu duck were lysed by addition of 0.83% ammonium chloride solution to the blood.Then the white blood cells were collected.The white blood cells wre sonicated to release the leukocytes granules, these granules were suspended in acetic and mixed overnight to extact the antimicrobial peptides.The solution containing the peptides was seperated from the granules,freezed-dried and the crude extraction of duck blood leukocyte antimicrobial peptides was obtained.Use the radial diffusion plate assay method,the crude extracts's antimicrobial activity against Escherichia.coli O78,Staphylococcus.aureus 1056MRSA and Candida.albicans ATCC10231 were revealed.The results showed that there wre antimicrobial matters in Yingtaogu duck leukocyte,and there was strong antimicrobial activity against all the bacteria strains above.Ion-exchange chromatography and reversed phase high performance liquid chromatography(RP-HPLC)were used to separated the components present in the crude extract.To determine whether the ion-exchange and RP-HPLC fractions displayed antimicrobial activity the radial diffusion plate assay method was used.The results showed that duck blood leukocyte antimicrobial peptides were cationic molecules.The antimicrobial activity of the ion-exchange chromatography fractions against Escherichia.coli 078 was stronger than that against Staphylococcus.aureus 1056MRSA and Candida.albicans ATCC10231.The antimicrobial activity of reversed phase high performance liquid chromatography(RP-HPLC)fractions against Staphylococcus.aureus 1056MRSA and Candida.albicans ATCC10231 were stronger than that against Escherichia.coli O78.Use MALDI-TOF-MS to determine molecular weight of some active components collected and separated by RP-HPLC.The results were 5351Da,5653 Da,5607 Da.5464 Da and 4325 Da. SeriesⅡEffects of crude extract from duck leukocyte on immunity of miceThe crude extract of duck leukocyte was obtained by using the method above.The animal experiment on the effects of crude extract from duck leukocyte on immunity of mice was designed into two groups(Group A and Group B),each group including 5 treatments:control group,protein control group,low dose group,middle dose group and high dose group.We used 12 mice in each treatment and used 120 mice in the whole experiment.The mice were feed timely in each day among the days.In Group A we sampled after 14 days and tested the immune apparatus index,E-rosette ratio and carbon clearance index(α).In Group B we sampled after 14 and 21 days and tested the immune apparatus index and T cell subsets of peripheral blood.The results showed that compared with control group,in high dose group the immune apparatus index reached a significant level(P＜0.05)and the carbon clearance index(α)reached a significant level(P＜0.01).The E-rosette ratio reached a significant level(P＜0.01)in crude extracts into stomach groups.And T cell subsets CD3~+,CD4~+ and CD4~+/CD8~+ reached a significant level(P＜0.01)in middle and high dose groups.So we can conclude that the immunity of mice could be enhanced by feeding duck crude extract.With feeding duck crude extract 14 and 21 days,the immune apparatus index,carbon clearance index(α),E-rosette ratio and T cell subsets CD3~+,CD4~+ and CD4~+ /CD8~+ reached a significant level(P＜0.05).But compared with after 14 days feeding,after 21 days feeding the groups didn't reach a significant level(P＞0.05).