鲜马乳乳清蛋白质组成及其抗氧化肽的分离纯化研究

发布时间：2018-05-24 19:37

本文选题：乳源性抗氧化肽 + 分离纯化　；参考：《新疆医科大学》2017年硕士论文

【摘要】：目的:采用柱色谱和SDS-PAGE电泳分离和鉴定马乳、牛乳、驴乳、驼乳乳清蛋白组成差异,并分离纯化鲜马乳乳源性生物活性肽,初步检测其生物活性,为鲜马乳功能研究提供依据。方法:(1)将四种鲜乳高速离心,分离乳清蛋白,采用离子交换层析和SDS-PAGE电泳方法,对四种不同鲜乳乳清蛋白组分进行鉴别,揭示其差异蛋白。(2)鲜马乳乳清蛋白酶解液分别用3Kd、10Kd、30Kd的超滤管进行富集并检测其抗氧化活性;选择活性较强的肽段采用Sephadex G-75和RP-HPLC进行纯化,MALDI-TOF/TOF进行结构鉴定,获得抗氧化活性肽。(3)采用MTT法测定鲜马乳抗氧化肽对人肺癌细胞系A549增殖的抑制作用,流式细胞仪检测其细胞周期和凋亡,通过检测GSH、SOD、MDA指标,揭示其氧抗氧化能力。结果:(1)鲜马乳蛋白与鲜牛乳蛋白组分相比较,发现其中存在三种已知蛋白,分子质量分别为69.60KD、46.22KD和26.35KD;与鲜驴乳蛋白组分相比较,发现其中存在三种已知蛋白,分子质量分别为100.92KD、69.60KD和76.31KD;与鲜驼乳蛋白组分相比较,发现其中存在四种已知蛋白,分子质量分别为100.92KD、95.56KD、26.35KD和76.31KD。(2)马乳乳清酶解物经过超滤、Sephadex G-75葡聚糖凝和半制备RP-HPLC分离纯化,MALDI-TOF/TOF质谱结构鉴定,初步确定抗氧化肽的分子量为1594.89KD,氨基酸组成为VAPFPQPVVPYPQR。(3)MTT法实验结果表明,抗氧化肽能抑制A549细胞增殖,将其阻滞在G1期,明显提高细胞凋亡,与模型组对比抗氧化肽高、中剂量组增加GSH含量和SOD的活力,降低MDA的分泌量,差异显著,具有统计学意义(P0.05)。结论:(1)通过检测发现,鲜马乳与驼乳、驴乳、牛乳乳清蛋白组成具有一定的差异性。(2)通过分离纯化和结构鉴定获得分子量为1594.89 KD,氨基酸组成为VAPFPQPVVPYPQR的鲜马乳乳源性抗氧化活性肽。(3)抗氧化肽抑制A549肺癌细胞增殖,增加GSH含量,提高SOD活力,降低MDA分泌量,具有一定的抗氧化活性。
[Abstract]:Objective: to separate and identify the difference of whey protein composition among horse milk, cow milk, donkey milk and camel milk by column chromatography and SDS-PAGE electrophoresis. Methods Whey protein was isolated from four kinds of fresh milk by high speed centrifugation. Four different components of whey protein were identified by ion exchange chromatography and SDS-PAGE electrophoresis. It was revealed that the milk whey proteolytic solution of fresh horse was enriched and its antioxidant activity was detected by ultrafiltration tube of 3KdD10KdDX 30Kd, and the structure of MALDI-TOF / TOF was identified by Sephadex G-75 and RP-HPLC. MTT assay was used to determine the inhibitory effect of fresh horse milk antioxidant peptide on the proliferation of human lung cancer cell line A549. Flow cytometry was used to detect cell cycle and apoptosis. Results compared with fresh milk protein, three known proteins were found, the molecular weight of which were 46.22KD and 26.35KDrespectively, and three known proteins were found compared with those of fresh donkey milk protein. The molecular weight was 100.92KD 69.60KD and 76.31KD, respectively. The molecular weight was 100.92 KDX 95.56 KD 26.35KD and 76.31KD. 2) the hydrolyzed whey of horse milk was identified by ultrafiltration Sephadex G-75 dextran coagulation and semi-prepared RP-HPLC. The molecular weight of antioxidant peptide was 1594.89KDand the amino acid composition was determined by VAPFPQPVVPYPQR.(3)MTT method, the molecular weight of the antioxidant peptide was 1594.89kDand the amino acid composition was determined by VAPFPQPVVPYPQR.(3)MTT method, and the molecular weight of MALDI-TOF / TOF was determined to be 1594.89kDand the amino acid composition was determined by VAPFPQPVVPYPQR.(3)MTT method. Antioxidative peptides could inhibit the proliferation of A549 cells and block them in G1 phase, which increased the apoptosis of A549 cells. Compared with the model group, antioxidant peptides increased the content of GSH and the activity of SOD, and decreased the secretion of MDA. It has statistical significance (P 0.05). ConclusionsConclusion 1) it was found that fresh horse milk, camel milk, donkey milk, The protein composition of whey protein was different. 2) the molecular weight was 1594.89 KD and the amino acid composition was VAPFPQPVVPYPQR. The antioxidant peptide inhibited the proliferation of A549 lung cancer cells and increased the content of GSH. Increasing the activity of SOD and decreasing the secretion of MDA, it has certain antioxidant activity.
【学位授予单位】：新疆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R284

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