固始鸡、丝毛乌鸡和鸵鸟中性粒细胞中抗菌肽分离纯化和部分性质研究
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摘要
本研究采集新鲜固始鸡、丝毛乌鸡和鸵鸟血液，用0.83％氯化铵反复裂解红细胞，得到白细胞；采用腹腔注射酪蛋白生理盐水的方法，从固始鸡和丝毛乌鸡腹水中得到白细胞。将收集的白细胞，通过超声波充分破碎、乙酸浸提、低温高速离心、旋转蒸发除去乙酸、冷冻干燥等方法，得到固始鸡血液(GX)、丝毛乌鸡血液(WX)、鸵鸟血液(TX)、固始鸡腹水(GF)和丝毛乌鸡腹水(WX)抗菌肽粗提物。将得到的各粗提物，经过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)进一步纯化后，收集各组分并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。
粗提物抑菌试验结果表明，在固始鸡、乌鸡和鸵鸟中性粒细胞中存在天然抗菌物质，并表现出对金黄色葡萄球菌(革兰氏阳性菌)、大肠杆菌(革兰氏阴性菌)和真菌(白色念珠菌)较强的抑制作用；从腹水中提取的抗菌肽比血液中的抗菌肽表现较强的抗菌活性；乌鸡中性粒细胞抗菌肽表现出比固始鸡和鸵鸟中性粒细胞抗菌肽强的抑制作用；
离子交换层析产物的抑菌试验结果表明，乌鸡、固始鸡和鸵鸟中性粒细胞中的抗菌物质是阳离子性质；和其它动物不同的是禽类阳离子抗菌肽主要作用于细菌，尤其是金黄色葡萄球菌(革兰氏阳性菌)，对白色念珠菌(真菌)的抑制作用较大肠杆菌(革兰氏阴性菌)强；取离子交换层析产物(有活性的峰2)，在沸水中处理5min～20min，在70℃～130℃处理30min，检测其对鸡大肠杆菌E.coli 078的抗菌活性。结果表明，固始鸡、乌鸡和鸵鸟中性粒细胞抗菌肽在100℃加热不同的时间，其抑菌圈直径没有明显的变化，在80～130℃条件下加热处理30min仍保持良好的抗菌活性，具有很好的热稳定性。
RP-HPLC纯化后抑菌试验结果表明，从固始鸡和乌鸡中性粒细胞中提取得到抗菌肽，有较强的抗白色念珠菌(真菌)和金黄色葡萄球菌(革兰氏阳性菌)的作用，而对大肠杆菌(革兰氏阴性菌)的抑制作用，则需要较大的浓度；固始鸡中性粒细胞抗菌肽对白色念珠菌(真菌)的抑制作用要大于黄色葡萄球菌(革兰氏阳性菌)，而乌鸡中性粒细胞抗菌肽对白色念珠菌(真菌)的抑制作用要小于黄色葡萄球菌(革兰氏阳性菌)；与已报道的鸵鸟中性粒细胞抗菌肽相比，本研究从得到的鸵鸟中性粒细胞抗菌肽显示出更强的抑菌活性；
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.
粗提物抑菌试验结果表明，在固始鸡、乌鸡和鸵鸟中性粒细胞中存在天然抗菌物质，并表现出对金黄色葡萄球菌(革兰氏阳性菌)、大肠杆菌(革兰氏阴性菌)和真菌(白色念珠菌)较强的抑制作用；从腹水中提取的抗菌肽比血液中的抗菌肽表现较强的抗菌活性；乌鸡中性粒细胞抗菌肽表现出比固始鸡和鸵鸟中性粒细胞抗菌肽强的抑制作用；
离子交换层析产物的抑菌试验结果表明，乌鸡、固始鸡和鸵鸟中性粒细胞中的抗菌物质是阳离子性质；和其它动物不同的是禽类阳离子抗菌肽主要作用于细菌，尤其是金黄色葡萄球菌(革兰氏阳性菌)，对白色念珠菌(真菌)的抑制作用较大肠杆菌(革兰氏阴性菌)强；取离子交换层析产物(有活性的峰2)，在沸水中处理5min～20min，在70℃～130℃处理30min，检测其对鸡大肠杆菌E.coli 078的抗菌活性。结果表明，固始鸡、乌鸡和鸵鸟中性粒细胞抗菌肽在100℃加热不同的时间，其抑菌圈直径没有明显的变化，在80～130℃条件下加热处理30min仍保持良好的抗菌活性，具有很好的热稳定性。
RP-HPLC纯化后抑菌试验结果表明，从固始鸡和乌鸡中性粒细胞中提取得到抗菌肽，有较强的抗白色念珠菌(真菌)和金黄色葡萄球菌(革兰氏阳性菌)的作用，而对大肠杆菌(革兰氏阴性菌)的抑制作用，则需要较大的浓度；固始鸡中性粒细胞抗菌肽对白色念珠菌(真菌)的抑制作用要大于黄色葡萄球菌(革兰氏阳性菌)，而乌鸡中性粒细胞抗菌肽对白色念珠菌(真菌)的抑制作用要小于黄色葡萄球菌(革兰氏阳性菌)；与已报道的鸵鸟中性粒细胞抗菌肽相比，本研究从得到的鸵鸟中性粒细胞抗菌肽显示出更强的抑菌活性；
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.
引文
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