苦荞过敏蛋白C端结构域的原核表达及其免疫学活性的鉴定

发布时间：2019-01-22 10:27

【摘要】：荞麦(Fagopyrum esculentum)作为一种功能性食物,越来越受到人们的广泛关注。它不仅具有丰富的营养价值,而且还有很高的药用价值。荞麦蛋白由较为独特的氨基酸组成。许多实验表明荞麦中的黄酮类物质含量较高,其产品具有明显的降低胆固醇和降血压等生物学作用,而且对糖尿病也有一定的疗效。然而,近年来的研究发现,荞麦中含有的过敏性成分通常可使接触或食用它的人产生过敏症状。日本和韩国学者先后从甜荞麦中分离获得16kDa、22-24 kDa、34-38 kDa及69 kDa等分子量不等的过敏蛋白。虽然对荞麦主要过敏原的确定还没有得到统一,各个研究小组的报道也存在一些差异,但一般认为22-24 kDa蛋白是荞麦中的主要过敏原。目前,国内外关于苦荞麦过敏的研究较少。本实验室曾以我国云南苦荞种子为原料,经分离纯化获得纯度均一的24 kDa天然蛋白,命名为TBa(tartary buckwheat allergen),通过免疫学检测发现其为苦荞中的主要过敏原。之后采用基因克隆法首次获得该苦荞过敏原的结构基因序列,并于2002年在GenBank登录(登录号为AY044918)。本实验是在已获得TBa结构基因的基础上,进行TBa的原核表达。首先将该基因克隆到原核表达载体pET-28a上,后将重组质粒转入大肠杆菌BL21(DE3)中进行表达。结果表达产物大部分以包涵体的形式存在,进一步经Ni~(2+)-NTA琼脂糖柱亲和纯化,得到纯度达95%以上的目的蛋白。用透析复性的方法将目的蛋白复性,复性产率达到约68%。Westernblot证实目的蛋白N端带有6个组氨酸标签。ELISA检测表明目的蛋白与IgE有特异性的结合,具有较高的免疫学活性。根据TBa的结构基因序列推导出该蛋白的氨基酸编码序列,应用Antigenic程序对推导出来的氨基酸序列进行抗原表位预测。初步的预测结果表明在成熟的TBa分子中包含八个抗原表位区段。选择含有抗原决定簇可能性最大的两个区段,根据其碱基序列,设计引物,克隆获得两个表位的基因片段。分别将两个基因片段连接到原核表达载体pET-32a上,并将重组质粒转入大肠杆菌BL21(DE3)中进行表达。对分离纯化后的表达产物进行免疫活性鉴定,初步表明两个推测的抗原表位区段均具有一定的抗体结合活性。
[Abstract]:As a functional food, buckwheat (Fagopyrum esculentum) has attracted more and more attention. It not only has rich nutrition value, but also has very high medicinal value. Buckwheat protein is composed of unique amino acids. Many experiments showed that the flavonoids content in buckwheat was high and its products had obvious biological effects such as lowering cholesterol and lowering blood pressure and also had certain curative effect on diabetes mellitus. However, recent studies have found that allergies in buckwheat often cause allergic symptoms in people who come into contact with or eat them. Japanese and Korean scholars isolated 16 kDa 22-24 kDa,34-38 kDa and 69 kDa respectively from buckwheat. Although the determination of the main allergens in buckwheat has not been unified and there are some differences in the reports of various research groups, it is generally believed that the 22-24 kDa protein is the main allergen in buckwheat. At present, there are few studies on hypersensitivity of Tartary buckwheat at home and abroad. The 24 kDa natural protein of Yunnan Tartary buckwheat seed was isolated and purified and named TBa (tartary buckwheat allergen), as the main allergen in Tartary buckwheat by immunological detection. The structural gene sequence of Tartary buckwheat allergen was obtained for the first time by gene cloning, and was registered on GenBank (accession number AY044918) in 2002. In this study, prokaryotic expression of TBa was carried out on the basis of obtained TBa structural genes. Firstly, the gene was cloned into prokaryotic expression vector pET-28a, then the recombinant plasmid was transferred into E. coli BL21 (DE3) for expression. Results most of the expressed products were in the form of inclusion bodies, and then purified by Ni~ (2)-NTA agarose column, the purity of the target protein was over 95%. The aim protein was renatured by dialysis renaturation, and the renaturation yield was about 68%.Westernblot. The N-terminal of the target protein had six histidine tags. ELISA analysis showed that the target protein had specific binding to IgE and had high immunological activity. According to the structural gene sequence of TBa, the amino acid coding sequence of the protein was deduced, and the deduced amino acid sequence was predicted by Antigenic program. Preliminary predictions indicate that mature TBa molecules contain eight antigenic epitopes. Two regions containing the most likely antigenic determinant were selected and primers were designed according to their base sequences to clone the two epitope gene fragments. The two gene fragments were ligated to the prokaryotic expression vector pET-32a and the recombinant plasmid was transformed into E. coli BL21 (DE3) for expression. The immunological activity of the expressed product was identified, and the results showed that the two hypothetical epitopes had a certain antibody binding activity.
【学位授予单位】：山西大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392;Q943.2;S517

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