| 摘要: |
| [目的]建立鸡白细胞中抗菌肽的分离纯化及活性鉴定方法.[方法]通过腹腔注射酪蛋白生理盐水,从鸡人工腹水中得到白细胞;将收集的白细胞,通过超声波反复破碎、体积分数1O%乙酸浸提、低温高速离心、旋转蒸发除去乙酸和冷冻干燥等方法,得到抗菌肽粗提物;将得到的粗提物,经CM-Sepharose Fast Flow弱酸性阳离子交换层析和反相高效液相色谱(RP-HPLC)纯化,收集分离的各组分,使用微量琼脂糖弥散法检测各组分的抗菌活性和最小抑菌浓度.[结果]共获得鸡抗菌肽粗提物0.270 g,经RP-HPLC分离纯化,抗菌肽离子交换阳性组分共分离出18个峰.其中6个峰对3种细菌均表现较强的抑菌活性,对鸡大肠杆菌E.coli 078(峰13)、金黄色葡萄球菌S.aureus 1056MRSA(峰15)和白色念珠菌C.albicans ATCC10231(峰15)的最小抑菌浓度分别为0.627,0.001和0.035 μg/mL.[结论]该法可以在实验室中用于鸡抗菌肽的提取及活性鉴定. |
| 关键词: 鸡白细胞 抗菌肽 纯化 活性检测 |
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| 基金项目:国家科技支撑计划项目（2006BDA01A09）；公益性行业（农业）科研专项（nyhyzx07-039）；河南省杰出人才创新基金项目（074200510009） |
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| Establishment of isolation, purification and activity identification method of chicken leucocyte antimicrobial peptides |
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| Abstract: |
| 【Objective】 The study was to extablish isolation,purification and activity identification method of chicken leucocyte antimicrobial peptides.【Method】 Sterile peritoneal exudates were harvested from chicken after injecting saline with 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.Iox exchange chromatography and reversed phase high performance liquid chromatography (RP-HPLC) were used to seperated the components present in the crude extract.Antimicrobial activity and the minimum inhibitory concentrations (MICs) were determined by the radial diffusion plate assay method.【Result】 0.270 g chicken crude extract was obtained.Using RP-HPLC the components in the active cationic fraction were separated into 18 peaks;peak 8 had good activity against all three test organisms;the minimum inhibitory concentrations of peak 13 against chicken E.coli O78,peak 15 against S.aureus 1056MRSA and peak 15 against C.albicans ATCC10231 was 0.627,0.001 and 0.035 μg/mL respectively.【Conclusion】 This method could be widely used to purify chicken antimicrobial peptides and identify antimicrobial activity in laboratory. |
| Key words: chicken leucocyte antimicrobial peptide purification activity identification |
