本文選題：乳源性抗氧化肽 + 分離純化��； 參考：《新疆醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:采用柱色譜和SDS-PAGE電泳分離和鑒定馬乳、牛乳、驢乳、駝乳乳清蛋白組成差異,并分離純化鮮馬乳乳源性生物活性肽,初步檢測其生物活性,為鮮馬乳功能研究提供依據(jù)。方法:(1)將四種鮮乳高速離心,分離乳清蛋白,采用離子交換層析和SDS-PAGE電泳方法,對四種不同鮮乳乳清蛋白組分進(jìn)行鑒別,揭示其差異蛋白。(2)鮮馬乳乳清蛋白酶解液分別用3Kd、10Kd、30Kd的超濾管進(jìn)行富集并檢測其抗氧化活性;選擇活性較強(qiáng)的肽段采用Sephadex G-75和RP-HPLC進(jìn)行純化,MALDI-TOF/TOF進(jìn)行結(jié)構(gòu)鑒定,獲得抗氧化活性肽。(3)采用MTT法測定鮮馬乳抗氧化肽對人肺癌細(xì)胞系A(chǔ)549增殖的抑制作用,流式細(xì)胞儀檢測其細(xì)胞周期和凋亡,通過檢測GSH、SOD、MDA指標(biāo),揭示其氧抗氧化能力。結(jié)果:(1)鮮馬乳蛋白與鮮牛乳蛋白組分相比較,發(fā)現(xiàn)其中存在三種已知蛋白,分子質(zhì)量分別為69.60KD、46.22KD和26.35KD;與鮮驢乳蛋白組分相比較,發(fā)現(xiàn)其中存在三種已知蛋白,分子質(zhì)量分別為100.92KD、69.60KD和76.31KD;與鮮駝乳蛋白組分相比較,發(fā)現(xiàn)其中存在四種已知蛋白,分子質(zhì)量分別為100.92KD、95.56KD、26.35KD和76.31KD。(2)馬乳乳清酶解物經(jīng)過超濾、Sephadex G-75葡聚糖凝和半制備RP-HPLC分離純化,MALDI-TOF/TOF質(zhì)譜結(jié)構(gòu)鑒定,初步確定抗氧化肽的分子量為1594.89KD,氨基酸組成為VAPFPQPVVPYPQR。(3)MTT法實(shí)驗(yàn)結(jié)果表明,抗氧化肽能抑制A549細(xì)胞增殖,將其阻滯在G1期,明顯提高細(xì)胞凋亡,與模型組對比抗氧化肽高、中劑量組增加GSH含量和SOD的活力,降低MDA的分泌量,差異顯著,具有統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論:(1)通過檢測發(fā)現(xiàn),鮮馬乳與駝乳、驢乳、牛乳乳清蛋白組成具有一定的差異性。(2)通過分離純化和結(jié)構(gòu)鑒定獲得分子量為1594.89 KD,氨基酸組成為VAPFPQPVVPYPQR的鮮馬乳乳源性抗氧化活性肽。(3)抗氧化肽抑制A549肺癌細(xì)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.
【學(xué)位授予單位】：新疆醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R284

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