本文關(guān)鍵詞：低聚糖糖基化法降低牛乳蛋白免疫原性的研究　出處：《北京林業(yè)大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： β-乳球蛋白 α_(s1)-酪蛋白 糖基化 抗原性 免疫原性

【摘要】： 糖基化法是降低食物過(guò)敏原蛋白免疫原性的有效方法之一,該方法是使致敏性食物蛋白通過(guò)在一定條件下的美拉德反應(yīng)和碳水化合物結(jié)合,使其抗原表位得到掩飾,從而降低過(guò)敏原蛋白的免疫原性。本研究選擇低聚半乳糖與低聚殼聚糖兩種低聚糖對(duì)牛乳中兩種重要的過(guò)敏原蛋白,p-乳球蛋白和αs1-酪蛋白進(jìn)行糖基化處理,通過(guò)動(dòng)物實(shí)驗(yàn)及ELISA試驗(yàn)等方法研究對(duì)該兩種乳蛋白的抗原性和免疫原性的影響。主要結(jié)果如下： 1)用低聚半乳糖與低聚殼聚糖在一定條件下通過(guò)美拉德反應(yīng)對(duì)牛乳p-乳球蛋白進(jìn)行糖基化處理,并用競(jìng)爭(zhēng)ELISA分析測(cè)定各糖基化產(chǎn)物的抗原性,結(jié)果表明抗原性均得到有效降低。用低聚半乳糖糖基化處理后的p-乳球蛋白糖基化產(chǎn)物的抗原抗體解離常數(shù)Kd較處理前上升5.8倍,低聚殼聚糖糖基化產(chǎn)物的解離常數(shù)Kd上升2.7倍,即用低聚半乳糖糖基化處理后,β-乳球蛋白的抗原性得到更有效降低。 2)通過(guò)動(dòng)物實(shí)驗(yàn)和ELISA試驗(yàn)檢測(cè)免疫原性的結(jié)果,分別用低聚半乳糖與低聚殼聚糖進(jìn)行糖基化處理后的牛乳p-乳球蛋白的免疫原性較處理前分別降低了18%與15%,低聚半乳糖糖基化法降低牛乳p-乳球蛋白免疫原性效果較理想。牛乳β-乳球蛋白進(jìn)行低聚半乳糖糖基化處理后,不僅免疫原性得到有效降低,誘導(dǎo)機(jī)體產(chǎn)生IgE的量減少15%。 3)等電點(diǎn)沉淀法從牛乳中提取酪蛋白,根據(jù)αs-酪蛋白的可溶于高濃度尿素溶液而不溶于低濃度尿素溶液以及對(duì)Ca2+敏感的特點(diǎn)從酪蛋白中提取αs-酪蛋白,通過(guò)陰離子交換層析法從αs-酪蛋白分離出αs1-酪蛋白。陰離子交換層析結(jié)果表明,用1mol/LNaCl作為洗脫液的進(jìn)行梯度洗脫時(shí),洗脫液體積分?jǐn)?shù)B%達(dá)到55%時(shí),as1-酪蛋白被洗脫出。用陰離子交換層析和SDS-PAGE凝膠電泳分析對(duì)αs1-酪蛋白組份進(jìn)行鑒定,結(jié)果表明αs1-酪蛋白得到較好的分離。 4)分別用低聚半乳糖與低聚殼聚糖對(duì)牛乳αs1-酪蛋白進(jìn)行糖基化處理,通過(guò)ELISA實(shí)驗(yàn)及動(dòng)物實(shí)驗(yàn)檢測(cè)其抗原性和免疫原性結(jié)果,兩種糖基化處理均能有效降低牛乳αs1-酪蛋白的抗原性和免疫原性。低聚半乳糖糖基化產(chǎn)物和低聚殼聚糖糖基化產(chǎn)物的抗原抗體結(jié)合常數(shù)Kd較αs1-酪蛋白分別上升3.7倍和2.5倍,即用低聚半乳糖糖基化處理后,牛乳αs1-酪蛋白的抗原性和免疫原性得到更有效降低。 以上結(jié)果表明,低聚半乳糖糖基化處理是降低牛乳p-乳球蛋白與αs1-酪蛋白的抗原性與免疫原性較有效方法。
[Abstract]:Glycosylation is one of the effective methods to reduce the immunogenicity of food allergen proteins. This method combines allergenic food proteins with carbohydrates through Maillard reaction under certain conditions. In order to reduce the immunogenicity of allergen proteins, oligosaccharides and oligosaccharides were selected as two important allergen proteins in milk. The antigenicity and immunogenicity of p- lactoglobulin and 偽 s1-casein were studied by animal experiment and ELISA test. The main results were as follows: 1) galactooligosaccharides and oligosaccharides were glycosylated by Maillard reaction under certain conditions, and the antigenicity of each glycosylation product was determined by competitive ELISA analysis. The results showed that the antigenicity was reduced effectively, and the dissociation constant of antigen and antibody of plactoglobulin glycosylation products treated with galactosylation was 5.8 times higher than that before treatment. The dissociation constant (KD) of oligochitosan glycosylation products increased 2.7 times, that is, the antigenicity of 尾 -lactoglobulin decreased more effectively after the treatment of oligogalactosylation. 2) the results of immunogenicity were detected by animal experiment and ELISA test. The immunogenicity of p- lactoglobulin after glycosylation of galactose and oligosaccharide decreased by 18% and 15% respectively. The effect of galactooligosylation to reduce the immunogenicity of milk p- lactoglobulin was ideal. The immunogenicity of milk 尾-lactoglobulin treated with galactooligosylated was decreased effectively. The amount that induces the body to produce IgE is reduced by 15%. 3) extraction of casein from milk by isoelectric precipitation. 偽 scasein was extracted from casein according to its solubility in high concentration urea solution but insoluble in low concentration urea solution and sensitive to Ca2. 偽 s 1-casein was isolated from 偽 scasein by anion exchange chromatography. The results of anion exchange chromatography showed that the gradient elution was carried out with 1mol / L NaCl as eluant. The as1-casein was washed out when the liquid integral number B% reached 55. The 偽 s1-casein component was identified by anion exchange chromatography and SDS-PAGE gel electrophoresis. The results showed that 偽 s 1 casein was separated well. 4) galactooligosaccharides and oligochitosan were used to glycosylation of milk 偽 s 1-casein, and the antigenicity and immunogenicity of 偽 s 1-casein were detected by ELISA and animal experiments. The antigenicity and immunogenicity of 偽 s 1-casein in milk were reduced by two kinds of glycosylation treatments, and the binding constant of antigen-antibody of galactooligosylated products and oligochitosan glycosylation products was higher than that of 偽 s 1-casein. The protein increased 3.7 and 2.5 times respectively. The antigenicity and immunogenicity of 偽 s 1-casein in milk decreased more effectively after galactooligosylation. The results showed that galactooligosylation was an effective method to reduce the antigenicity and immunogenicity of p- lactoglobulin and 偽 s _ 1-casein.
【學(xué)位授予單位】：北京林業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R392

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