过敏原Fag t 1 低变应原分子的构建及重组研究

发布时间：2018-01-06 04:19

本文关键词：过敏原Fag t 1 低变应原分子的构建及重组研究　出处：《山西大学》2012年硕士论文　论文类型：学位论文

【摘要】：苦荞作为一种药食同源的作物,具有很好的营养价值和保健作用。但是有报道苦荞可能引起过敏症状,如哮喘、皮炎等,而苦荞种子中的储藏蛋白被鉴定为苦荞主要过敏原。该蛋白属于Cupin家族的11S球蛋白,在苦荞种子中含量丰富,稳定性好。本文主要从基因工程角度和食品加工角度对苦荞过敏原进行了研究。本论文首先采用基因重组技术对苦荞过敏原Fag t1的核苷酸序列进行了重新构建,并将重组分子在原核载体中表达获得几种重组蛋白,分别命名为Fag t1-rs1, Fagt1-rs2,和Fagt1-rs3。这些重组蛋白由于失去天然蛋白特殊的结构而丧失了IgE结合活性,因而免疫活性显著降低。相比天然Fag t1,重组分子免疫活性降低了约90%,这些低敏分子有希望成为过敏特异性免疫治疗的疫苗。同时,解聚状态的Fagt1重组分子抗蛋白酶活性也明显下降。这些结果说明,11S种子储藏蛋白的抗酶解稳定性和免疫活性都依赖于其空间结构的完整性,设计实验改造Cupin食物过敏原的天然构象是降低其变应原性的有效策略。其次,本论文还就食品加工过程对苦荞过敏原免疫活性的影响进行了研究。通过提取苦荞自身的多糖,研究在多糖成分存在下,过敏蛋白发生的美拉德反应。结果表明,在70℃,3d条件下过敏蛋白与糖自发美拉德共价连接后,糖化蛋白在许多性质诸如,电泳性质,二级结构,溶解度及稳定性等都发生了改变。同时通过ELISA和免疫点杂交,使用患者血清和兔多抗对其免疫活性进行分析,结果显示糖化蛋白IgE和IgG结合活性相比天然蛋白明显降低。我们推测过敏原与糖分子的结合改变了蛋白质的结构,尤其抗原表位发生了变化,导致过敏原活性明显降低。本实验结果提示,食品加工中的加热糖化过程可能会影响过敏活性。
[Abstract]:Buckwheat as a medicinal and edible crops, has good nutritional value and health care function. But there are reports of buckwheat may cause allergic symptoms such as asthma, dermatitis, and buckwheat seeds in storage protein was identified as the major allergen in tartary buckwheat. The 11S globulin protein belonging to the Cupin family, rich in content. Tartary buckwheat has good stability. This paper mainly from the perspective of genetic engineering and food processing of buckwheat allergens were studied.
Firstly, using the gene recombination technique of nucleotide sequence of tartary buckwheat allergen Fag T1 was re constructed, and the recombinant expression of several recombinant proteins in prokaryotic vectors, named Fag t1-rs1, Fagt1-rs2 Fagt1-rs3., and the recombinant protein to natural protein structure due to special and lost IgE binding activity. Thus the immune activity decreased significantly. Compared with natural Fag T1, the immune activity of recombinant molecules is reduced by about 90%, these molecules have low sensitivity to become allergy specific immunotherapy vaccine. At the same time, the recombinant Fagt1 molecular depolymerization state of anti protease activity also decreased significantly. These results suggest that the integrity of the anti enzyme stability and immune activity depending on the spatial structure of the seed storage protein 11S, Cupin transformation design experiment of food allergens is to reduce the natural conformation of effective strategies of its allergenicity.
Secondly, this paper also studies the effect of food processing on buckwheat allergen activity. By extracting polysaccharide from Tartary Buckwheat's research in the presence of polysaccharides, Maillard reaction allergic protein occurred. The results showed that at 70 degrees C, allergic protein and sugar under the condition of 3D spontaneous Maillard covalently linked. Electrophoretic properties of glycated protein in many properties, such as, two level structure, solubility and stability are changed. At the same time by ELISA and immuno dot blot, using serum and rabbit polyclonal antibodies were analyzed on the immune activity, the results showed IgE glycosylated protein and IgG binding activity than natural protein decreased significantly. We speculate that the combination of allergy the original and the sugar molecule changes the structure of the protein, especially epitope changes, resulting in allergen activity decreased significantly. The results suggest that the heat in the saccharification of food processing The process may affect the anaphylaxis.

【学位授予单位】：山西大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392

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