本文關(guān)鍵詞： 牛奶過敏 β-乳球蛋白 特異性IgE 抗原表位 重組表達　出處：《天津醫(yī)科大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:血清特異性IgE是診斷食物過敏的重要手段,標(biāo)準(zhǔn)化已知抗原是研制體外特異性IgE診斷試劑的重要前提,制備食物致敏組分的抗原表位重組蛋白是解決抗原標(biāo)準(zhǔn)化的重要措施。本研究以牛奶中最重要的過敏原“β-乳球蛋白”為例,從分析其抗原表位入手,分析重要抗原表位與患者血清sIgE的反應(yīng)頻率,從而篩選出其關(guān)鍵抗原表位并構(gòu)建關(guān)鍵抗原表位重組體,嘗試采用抗原表位重組體替代天然蛋白的可能性。方法:(1)β-乳球蛋白關(guān)鍵抗原表位篩選及其血清異質(zhì)性分析:首先在NCBI數(shù)據(jù)庫查得牛奶β-乳球蛋白的氨基酸序列(162個氨基酸),用末端重疊的方法合成β-乳球蛋白的多肽,然后用斑點印跡的方法,以20例牛奶過敏患者血清中的特異性IgE作為一抗,觀察多肽與牛奶過敏患者的反應(yīng)頻率有無差異及不同個體間所識別的多肽有無差異。(2)β-乳球蛋白關(guān)鍵抗原表位串聯(lián)重組體的構(gòu)建及其初步應(yīng)用研究:將反應(yīng)頻率最高的3個多肽進行串聯(lián)表達,相鄰兩個多肽之間用一個甘氨酸做接頭,首先用DNAStar生物信息學(xué)軟件對3個表位的6種組合方式進行預(yù)測,選出最佳組合方式;委托上海生工生物有限公司合成關(guān)鍵抗原表位串聯(lián)體的DNA序列,并與pET-42a(+)質(zhì)粒載體進行連接,將構(gòu)建好的質(zhì)粒轉(zhuǎn)化到BL21(DE3)感受態(tài)細胞中,用IPTG誘導(dǎo)劑進行誘導(dǎo)表達,最后利用鎳柱純化出單一組分的關(guān)鍵抗原表位串聯(lián)體重組蛋白,最后采用western-blot、斑點印跡方法和活性氧傳遞均相發(fā)光免疫測定技術(shù)鑒定重組蛋白的免疫學(xué)活性。結(jié)果:(1)患者血清中sIgE所識別抗原表位的異質(zhì)性分析:在20例牛奶過敏患者血清的斑點印跡結(jié)果中有一部分血清所識別的多肽相同,如1號、5號、6號、11號牛奶過敏患者血清所識別的表位均為1號多肽,有一部分牛奶過敏患者血清所識別的表位均不完全相同。根據(jù)多肽反應(yīng)頻率的不同,篩選出β-乳球蛋白最主要表位的氨基酸序列為AA76-95、AA1-20、AA106-125,主要表位的氨基酸序列為AA91-110、AA61-81、AA121-140,次要表位的氨基酸序列為AA 46-65、AA 31-50、AA 151-162。(2)β-乳球蛋白關(guān)鍵抗原表位串聯(lián)體重組蛋白的表達及鑒定:通過DNAStar軟件對理論上6種組合方式進行了分析,得到最佳組合方式,成功構(gòu)建了大腸桿菌原核表達載體,IPTG誘導(dǎo)表達后,獲得大小為36kDa左右的融合蛋白,并純化得到單一組分的36kDa大小的重組蛋白,并經(jīng)免疫印跡法、斑點印跡法鑒定其免疫活性,并利用重組蛋白作為檢測抗原建立了一種活性氧傳遞均相發(fā)光免疫測定技術(shù)來檢測血清sIgE。結(jié)論:本研究對牛奶主要過敏原β-乳球蛋白的抗原表位進行了解析,不同牛奶過敏患者血清所識別的抗原表位具有異質(zhì)性。成功表達了β-乳球蛋白關(guān)鍵抗原表位串聯(lián)體重組蛋白,并鑒定了關(guān)鍵抗原串聯(lián)體重組蛋白具有良好的免疫學(xué)活性,在檢測牛奶過敏疾病中有可能代替天然抗原,為新一代診斷試劑的制備奠定基礎(chǔ)。
[Abstract]:Objective: serum specific IgE is an important method for the diagnosis of food allergy, and standardized known antigen is an important prerequisite for the development of in vitro specific IgE diagnostic reagent. The preparation of antigenic epitope recombinant protein of food sensitized component is an important measure to solve the problem of antigen standardization. In this study, the most important allergen "尾 -lactoglobulin" in milk was taken as an example to analyze the antigen epitopes of 尾 -lactoglobulin (尾 -lactoglobulin). The reaction frequency between the important antigen epitope and the patient's serum sIgE was analyzed, and the key antigen epitopes were screened out and the recombination of the key antigen epitopes was constructed. Methods the screening of 尾 -lactoglobulin epitopes and the analysis of serum heterogeneity: first, the amino acid sequence of milk 尾 -lactoglobulin was identified by NCBI database and the amino acid sequence of milk 尾 -lactoglobulin was obtained. The peptide of 尾-lactoglobulin was synthesized by the method of terminal overlap. Then the specific IgE in the sera of 20 patients with milk allergy was used as the first antibody by dot blot. Study on the construction of 尾 -lactoglobulin key antigen epitope tandem recombinant and its preliminary application study on the difference of the frequency of reaction between polypeptide and milk allergy patients and the difference of polypeptide recognized by different individuals. Three high peptides were expressed in tandem. One glycine was used as the junction between the two adjacent peptides. First, the six combinations of the three epitopes were predicted by DNAStar bioinformatics software, and the best combination was selected. The DNA sequence of the key antigen epitope was synthesized by Shanghai Biotechnology Co., Ltd., and ligated with pET-42a () plasmid vector. The constructed plasmid was transformed into BL21DE-3) competent cells, and was induced to express by IPTG inducer. Finally, the key epitopes of single component were purified by nickel column. Finally, the immunological activity of the recombinant protein was identified by Western blot, dot blotting and reactive oxygen species transfer homogenous luminescence immunoassay. Results the heterogeneity of antigen epitopes recognized by sIgE in serum of 20 cases of milk hypersensitivity was analyzed. Some of the sera recognized the same polypeptide in the blotting results of the patient's serum. For example, the epitopes recognized in the sera of milk allergy patients 1, 5, 6 and 11 are all polypeptide 1, and the epitopes recognized by some milk allergy patients are not identical, depending on the frequency of polypeptide reaction. The main amino acid sequence of 尾 -lactoglobulin was AA76-95A1-20A106-125, the main epitope was AA91-110A61-81AA-121-140, and the secondary epitope was AA46-65A31-50AA151-162.2). In this paper, six kinds of theoretical combination methods are analyzed by DNAStar software. The best combination method was obtained. After the expression of E. coli prokaryotic expression vector was induced by IPTG, the fusion protein of about 36kDa was obtained, and the recombinant protein with the size of 36kDa of a single component was purified, and the recombinant protein was purified by Western blotting. The immunological activity was identified by dot blot. Using recombinant protein as antigen, a reactive oxygen species transfer homogenous luminescence immunoassay (Ros) was developed to detect serum Siga. Conclusion: the epitopes of 尾-lactoglobulin, the main allergen of milk, were analyzed in this study. The antigenic epitopes recognized by serum of different milk allergy patients were heterogeneity. 尾 -lactoglobulin key antigen epitope tandem weight histone was successfully expressed, and the key antigen tandem weight histone was identified as having good immunological activity. It is possible to replace the natural antigen in the detection of milk allergy diseases, which will lay a foundation for the preparation of a new generation of diagnostic reagents.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R446.6

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