鸭白细胞抗菌肽的初步鉴定及粗提物对小鼠免疫功能的影响
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摘要
本试验以樱桃谷鸭为研究对象,对其血液白细胞中抗菌肽成分进行了提取和分离,并初步研究了其对小鼠免疫功能的影响。本试验共分为两个系列。
系列Ⅰ鸭白细胞抗菌肽的提取、分离与初步鉴定
采集新鲜樱桃谷鸭血液,用0.83%氯化铵反复裂解红细胞,得到白细胞,将收集到的白细胞,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,得到鸭白细胞抗菌肽粗提物。使用微量琼脂糖弥散法测定其抑菌活性,结果表明,在樱桃谷鸭血液白细胞中存在天然抗菌物质,并表现出对革兰氏阳性菌(金黄色葡萄球菌),革兰氏阴性菌(大肠杆菌)和白色念珠菌(真菌)较强的抑制作用。
将得到的粗提物,经过CM-Sepharose Fast Flow阳离子交换层析和反相高效液相色谱法(RP-HPLC)纯化后,收集各组分,并使用微量琼脂糖弥散法测定各组分的抑菌活性。抑菌试验显示,抗菌物质是阳离子性质,对革兰氏阴性菌(大肠杆菌)的抑制作用稍强于对革兰氏阳性菌(金黄色葡萄球菌)和白色念珠菌(真菌)的抑制作用。而经过RP-HPLC分离纯化后显示,提取到的抗菌肽有较强的抑制革兰氏阳性菌(金黄色葡萄球菌)和白色念珠菌(真菌)生长的作用,而对革兰氏阴性菌(大肠杆菌)的抑制作用较弱,
使用MALDI-TOF-MS法对经RP-HPLC纯化后收集的几个活性组分进行分子量的测定,其结果分别为5351Da、5653 Da、5607 Da、5464 Da和4325 Da。
系列Ⅱ鸭白细胞粗提物对小鼠免疫功能的影响
采取上述方法制备鸭血液白细胞粗提物。粗提物对小鼠免疫功能影响的动物试验分A、B两组。A、B组各分为五组:对照组、蛋白对照组、低剂量组、中剂量组和高剂量组。每组12只小鼠。每日定时灌胃,A组灌胃14天后采样,测定小鼠免疫器官指数、E玫瑰花环形成率、碳粒廓清指数。B组灌胃14天或21天后采样,测定小鼠免疫器官指数、外周血T细胞亚群。测定结果表明:白细胞粗提物灌胃不同的剂量,与对照处理相比,高剂量组的免疫器官指数差异显著(P＜0.05),巨噬细胞吞噬指数差异极显著(P＜0.01);各剂量组的E玫瑰花环形成率均有极显著差异(P＜0.01);中、高剂量组的T细胞亚群CD3~+、CD4~+和CD4~+/CD8~+差异极显著(P＜0.01),初步说明白细胞粗提物能提高小鼠的机体免疫力。白细胞粗提物灌胃14天和21天后,小鼠的免疫器官指数、巨噬细胞吞噬指数、E玫瑰花环形成率和T细胞亚群CD3~+、CD4~+和CD4~+/CD8~+均有显著提高,但灌胃14天的各组与灌胃21天的各组相比,差异不显著(P＞0.05)。
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 leukocyte
The 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 mice
The 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).
系列Ⅰ鸭白细胞抗菌肽的提取、分离与初步鉴定
采集新鲜樱桃谷鸭血液,用0.83%氯化铵反复裂解红细胞,得到白细胞,将收集到的白细胞,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,得到鸭白细胞抗菌肽粗提物。使用微量琼脂糖弥散法测定其抑菌活性,结果表明,在樱桃谷鸭血液白细胞中存在天然抗菌物质,并表现出对革兰氏阳性菌(金黄色葡萄球菌),革兰氏阴性菌(大肠杆菌)和白色念珠菌(真菌)较强的抑制作用。
将得到的粗提物,经过CM-Sepharose Fast Flow阳离子交换层析和反相高效液相色谱法(RP-HPLC)纯化后,收集各组分,并使用微量琼脂糖弥散法测定各组分的抑菌活性。抑菌试验显示,抗菌物质是阳离子性质,对革兰氏阴性菌(大肠杆菌)的抑制作用稍强于对革兰氏阳性菌(金黄色葡萄球菌)和白色念珠菌(真菌)的抑制作用。而经过RP-HPLC分离纯化后显示,提取到的抗菌肽有较强的抑制革兰氏阳性菌(金黄色葡萄球菌)和白色念珠菌(真菌)生长的作用,而对革兰氏阴性菌(大肠杆菌)的抑制作用较弱,
使用MALDI-TOF-MS法对经RP-HPLC纯化后收集的几个活性组分进行分子量的测定,其结果分别为5351Da、5653 Da、5607 Da、5464 Da和4325 Da。
系列Ⅱ鸭白细胞粗提物对小鼠免疫功能的影响
采取上述方法制备鸭血液白细胞粗提物。粗提物对小鼠免疫功能影响的动物试验分A、B两组。A、B组各分为五组:对照组、蛋白对照组、低剂量组、中剂量组和高剂量组。每组12只小鼠。每日定时灌胃,A组灌胃14天后采样,测定小鼠免疫器官指数、E玫瑰花环形成率、碳粒廓清指数。B组灌胃14天或21天后采样,测定小鼠免疫器官指数、外周血T细胞亚群。测定结果表明:白细胞粗提物灌胃不同的剂量,与对照处理相比,高剂量组的免疫器官指数差异显著(P＜0.05),巨噬细胞吞噬指数差异极显著(P＜0.01);各剂量组的E玫瑰花环形成率均有极显著差异(P＜0.01);中、高剂量组的T细胞亚群CD3~+、CD4~+和CD4~+/CD8~+差异极显著(P＜0.01),初步说明白细胞粗提物能提高小鼠的机体免疫力。白细胞粗提物灌胃14天和21天后,小鼠的免疫器官指数、巨噬细胞吞噬指数、E玫瑰花环形成率和T细胞亚群CD3~+、CD4~+和CD4~+/CD8~+均有显著提高,但灌胃14天的各组与灌胃21天的各组相比,差异不显著(P＞0.05)。
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 leukocyte
The 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 mice
The 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).
引文
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