Studies On Isolation, Purification And Partial Characters Of Gushi Chicken, Wu Chicken And Ostrich Neutrophil Antimicrobial Peptides

The fresh red blood cells of Gushi chicken, Wu chicken and ostrich were lysed by addition of 0.83% ammonium chlorid solution to the blood. Sterile peritoneal exudates were harvested from Gushi chicken and Wu chicken 6h after injecting saline witn starch, then the white blood cells were collected. The white blood cells were sonicated to release the neutrophil granules, these granules were suspended in acetic acid and mixed overnight to extract the antimicrobial peptides. The solution containing the peptides was seperated from the granules, freeze-dried and the crude extraction of Gushi chicken blood neutrophil antimicrobial peptides, Wu chicken blood neutrophil antimicrobial peptides, ostrich blood neutrophil antimicrobial peptides, Gushi chicken peritoneal neutrophil antimicrobial peptides and Wu chicken peritoneal neutrophil antimicrobial peptides were obtained. Iox-exchange chromatography and reversed phase high performance liquid chromatography (RP-HPLC) were used to seperated the components present in the crude extract. To determine whether the ion-exchange and RP-HPLC fractions displayed antimicrobial activity and the minimum inhibitory concentrations (MICs) the radial diffusion plate assay method was used.Use the radial diffusion plate assay method, the crude extracts' antimicrobial activity against Escherichia.coli 078, Staphylococcus.aureus 1056MRSA and Candida.albicans ATCC10231 were revealed. The results showed that there were antimicrobial matters in Gushi chicken, Wu chicken and ostrich neutrophil, and there were strongly active against all the bacteria strains above. The antimicrobial activity of antimicrobial peptides in peritoneal were stronger than that in blood, the antimicrobial activity of Wu chicken neutrophil antimicrobial peptides were stronger than Gushi chicken and ostrich neutrophil antimicrobial peptides.Use the radial diffusion plate assay method, the iox-exchange chromatography fractions' antimicrobial activity against Escherichia.coli 078, Staphylococcus.aureus 1056MRSA and Candida.albicans ATCC10231 were revealed. The results showed that Gushi chicken, Wu chicken and ostrich neutrophil antimicrobial peptides were cationic molecules. Different to other animals the avian cationic antimicrobial peptides were active against the bacteria, especially the Staphylococcus.aureus 1056MRSA, and the antimicrobial activity of the iox-exchange chromatography fractions against the Candida.albicans ATCC10231 were stronger than that against Escherichia.coli O78. The iox-exchange chromatography fractions (the active fraction 2) were dealed 5～20min at 100℃and 30min at 70℃～130℃, use the radial diffusion plate assay method, antimicrobial activity against Escherichia.coli O78 was tested.. The results showed that the clear zone of Gushi chicken, Wu chicken and ostrich neutrophil antimicrobial peptides had no distinctly changes at 100℃in different time, and the antimicrobial activity was still strong when the fractions were treated 30min at 70～130℃. The results indicated that the antimicrobial peptides have good character of hot stabilization.Use the radial diffusion plate assay method, the reversed phase high performance liquid chromatography (RP-HPLC) fractions' antimicrobial activity against Escherichia.coli O78, Staphylococcus.aureus 1056MRSA and Candida.albicans ATCC10231 were revealed. The results showed that Gushi chicken and Wu chicken neutrophil antimicrobial peptides had stronger activity against Staphylococcus.aureus 1056MRSA and Candida.albicans ATCC10231, but the activity against Escherichia.coli O78 need high concentration. The antimicrobial activity of Gushi chicken neutrophil antimicrobial peptides against Candida.albicans ATCC10231 were stronger than that of Staphylococcus.aureus 1056MRSA. But the antimicrobial activity of Wu chicken neutrophil antimicrobial peptides against Candida.albicans ATCC10231 were weaker than that of Staphylococcus.aureus 1056MRSA. Compared with the reported ostrich neutrophil antimicrobial peptides, the peptides of our research obtained have stronger activity.