低聚糖糖基化法降低牛乳蛋白免疫原性的研究

发布时间：2018-01-07 02:42

本文关键词：低聚糖糖基化法降低牛乳蛋白免疫原性的研究　出处：《北京林业大学》2010年硕士论文　论文类型：学位论文

【摘要】： 糖基化法是降低食物过敏原蛋白免疫原性的有效方法之一,该方法是使致敏性食物蛋白通过在一定条件下的美拉德反应和碳水化合物结合,使其抗原表位得到掩饰,从而降低过敏原蛋白的免疫原性。本研究选择低聚半乳糖与低聚壳聚糖两种低聚糖对牛乳中两种重要的过敏原蛋白,p-乳球蛋白和αs1-酪蛋白进行糖基化处理,通过动物实验及ELISA试验等方法研究对该两种乳蛋白的抗原性和免疫原性的影响。主要结果如下： 1)用低聚半乳糖与低聚壳聚糖在一定条件下通过美拉德反应对牛乳p-乳球蛋白进行糖基化处理,并用竞争ELISA分析测定各糖基化产物的抗原性,结果表明抗原性均得到有效降低。用低聚半乳糖糖基化处理后的p-乳球蛋白糖基化产物的抗原抗体解离常数Kd较处理前上升5.8倍,低聚壳聚糖糖基化产物的解离常数Kd上升2.7倍,即用低聚半乳糖糖基化处理后,β-乳球蛋白的抗原性得到更有效降低。 2)通过动物实验和ELISA试验检测免疫原性的结果,分别用低聚半乳糖与低聚壳聚糖进行糖基化处理后的牛乳p-乳球蛋白的免疫原性较处理前分别降低了18%与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较理想。牛乳β-乳球蛋白进行低聚半乳糖糖基化处理后,不仅免疫原性得到有效降低,诱导机体产生IgE的量减少15%。 3)等电点沉淀法从牛乳中提取酪蛋白,根据αs-酪蛋白的可溶于高浓度尿素溶液而不溶于低浓度尿素溶液以及对Ca2+敏感的特点从酪蛋白中提取αs-酪蛋白,通过阴离子交换层析法从αs-酪蛋白分离出αs1-酪蛋白。阴离子交换层析结果表明,用1mol/LNaCl作为洗脱液的进行梯度洗脱时,洗脱液体积分数B%达到55%时,as1-酪蛋白被洗脱出。用阴离子交换层析和SDS-PAGE凝胶电泳分析对αs1-酪蛋白组份进行鉴定,结果表明αs1-酪蛋白得到较好的分离。 4)分别用低聚半乳糖与低聚壳聚糖对牛乳αs1-酪蛋白进行糖基化处理,通过ELISA实验及动物实验检测其抗原性和免疫原性结果,两种糖基化处理均能有效降低牛乳αs1-酪蛋白的抗原性和免疫原性。低聚半乳糖糖基化产物和低聚壳聚糖糖基化产物的抗原抗体结合常数Kd较αs1-酪蛋白分别上升3.7倍和2.5倍,即用低聚半乳糖糖基化处理后,牛乳αs1-酪蛋白的抗原性和免疫原性得到更有效降低。以上结果表明,低聚半乳糖糖基化处理是降低牛乳p-乳球蛋白与αs1-酪蛋白的抗原性与免疫原性较有效方法。
[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 伪 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. 伪 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. 伪 s 1-casein was isolated from 伪 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 伪 s1-casein component was identified by anion exchange chromatography and SDS-PAGE gel electrophoresis. The results showed that 伪 s 1 casein was separated well. 4) galactooligosaccharides and oligochitosan were used to glycosylation of milk 伪 s 1-casein, and the antigenicity and immunogenicity of 伪 s 1-casein were detected by ELISA and animal experiments. The antigenicity and immunogenicity of 伪 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 伪 s 1-casein. The protein increased 3.7 and 2.5 times respectively. The antigenicity and immunogenicity of 伪 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 伪 s _ 1-casein.
【学位授予单位】：北京林业大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

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