【摘要】：蕎麥(Fagopyrum esculentum)作為一種功能性食物,越來(lái)越受到人們的廣泛關(guān)注。它不僅具有豐富的營(yíng)養(yǎng)價(jià)值,而且還有很高的藥用價(jià)值。蕎麥蛋白由較為獨(dú)特的氨基酸組成。許多實(shí)驗(yàn)表明蕎麥中的黃酮類(lèi)物質(zhì)含量較高,其產(chǎn)品具有明顯的降低膽固醇和降血壓等生物學(xué)作用,而且對(duì)糖尿病也有一定的療效。然而,近年來(lái)的研究發(fā)現(xiàn),蕎麥中含有的過(guò)敏性成分通�？墒菇佑|或食用它的人產(chǎn)生過(guò)敏癥狀。日本和韓國(guó)學(xué)者先后從甜蕎麥中分離獲得16kDa、22-24 kDa、34-38 kDa及69 kDa等分子量不等的過(guò)敏蛋白。雖然對(duì)蕎麥主要過(guò)敏原的確定還沒(méi)有得到統(tǒng)一,各個(gè)研究小組的報(bào)道也存在一些差異,但一般認(rèn)為22-24 kDa蛋白是蕎麥中的主要過(guò)敏原。目前,國(guó)內(nèi)外關(guān)于苦蕎麥過(guò)敏的研究較少。本實(shí)驗(yàn)室曾以我國(guó)云南苦蕎種子為原料,經(jīng)分離純化獲得純度均一的24 kDa天然蛋白,命名為T(mén)Ba(tartary buckwheat allergen),通過(guò)免疫學(xué)檢測(cè)發(fā)現(xiàn)其為苦蕎中的主要過(guò)敏原。之后采用基因克隆法首次獲得該苦蕎過(guò)敏原的結(jié)構(gòu)基因序列,并于2002年在GenBank登錄(登錄號(hào)為AY044918)。 本實(shí)驗(yàn)是在已獲得TBa結(jié)構(gòu)基因的基礎(chǔ)上,進(jìn)行TBa的原核表達(dá)。首先將該基因克隆到原核表達(dá)載體pET-28a上,后將重組質(zhì)粒轉(zhuǎn)入大腸桿菌BL21(DE3)中進(jìn)行表達(dá)。結(jié)果表達(dá)產(chǎn)物大部分以包涵體的形式存在,進(jìn)一步經(jīng)Ni~(2+)-NTA瓊脂糖柱親和純化,得到純度達(dá)95%以上的目的蛋白。用透析復(fù)性的方法將目的蛋白復(fù)性,復(fù)性產(chǎn)率達(dá)到約68%。Westernblot證實(shí)目的蛋白N端帶有6個(gè)組氨酸標(biāo)簽。ELISA檢測(cè)表明目的蛋白與IgE有特異性的結(jié)合,具有較高的免疫學(xué)活性。 根據(jù)TBa的結(jié)構(gòu)基因序列推導(dǎo)出該蛋白的氨基酸編碼序列,應(yīng)用Antigenic程序?qū)ν茖?dǎo)出來(lái)的氨基酸序列進(jìn)行抗原表位預(yù)測(cè)。初步的預(yù)測(cè)結(jié)果表明在成熟的TBa分子中包含八個(gè)抗原表位區(qū)段。選擇含有抗原決定簇可能性最大的兩個(gè)區(qū)段,根據(jù)其堿基序列,設(shè)計(jì)引物,克隆獲得兩個(gè)表位的基因片段。分別將兩個(gè)基因片段連接到原核表達(dá)載體pET-32a上,并將重組質(zhì)粒轉(zhuǎn)入大腸桿菌BL21(DE3)中進(jìn)行表達(dá)。對(duì)分離純化后的表達(dá)產(chǎn)物進(jìn)行免疫活性鑒定,初步表明兩個(gè)推測(cè)的抗原表位區(qū)段均具有一定的抗體結(jié)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.
【學(xué)位授予單位】：山西大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2006
【分類(lèi)號(hào)】：R392;Q943.2;S517

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