β-乳球蛋白抗原表位谱的解析及关键抗原表位串联体的重组表达与鉴定

发布时间：2018-02-11 11:16

本文关键词： 牛奶过敏 β-乳球蛋白特异性IgE 抗原表位重组表达　出处：《天津医科大学》2015年硕士论文　论文类型：学位论文

【摘要】：目的:血清特异性IgE是诊断食物过敏的重要手段,标准化已知抗原是研制体外特异性IgE诊断试剂的重要前提,制备食物致敏组分的抗原表位重组蛋白是解决抗原标准化的重要措施。本研究以牛奶中最重要的过敏原“β-乳球蛋白”为例,从分析其抗原表位入手,分析重要抗原表位与患者血清sIgE的反应频率,从而筛选出其关键抗原表位并构建关键抗原表位重组体,尝试采用抗原表位重组体替代天然蛋白的可能性。方法:(1)β-乳球蛋白关键抗原表位筛选及其血清异质性分析:首先在NCBI数据库查得牛奶β-乳球蛋白的氨基酸序列(162个氨基酸),用末端重叠的方法合成β-乳球蛋白的多肽,然后用斑点印迹的方法,以20例牛奶过敏患者血清中的特异性IgE作为一抗,观察多肽与牛奶过敏患者的反应频率有无差异及不同个体间所识别的多肽有无差异。(2)β-乳球蛋白关键抗原表位串联重组体的构建及其初步应用研究:将反应频率最高的3个多肽进行串联表达,相邻两个多肽之间用一个甘氨酸做接头,首先用DNAStar生物信息学软件对3个表位的6种组合方式进行预测,选出最佳组合方式;委托上海生工生物有限公司合成关键抗原表位串联体的DNA序列,并与pET-42a(+)质粒载体进行连接,将构建好的质粒转化到BL21(DE3)感受态细胞中,用IPTG诱导剂进行诱导表达,最后利用镍柱纯化出单一组分的关键抗原表位串联体重组蛋白,最后采用western-blot、斑点印迹方法和活性氧传递均相发光免疫测定技术鉴定重组蛋白的免疫学活性。结果:(1)患者血清中sIgE所识别抗原表位的异质性分析:在20例牛奶过敏患者血清的斑点印迹结果中有一部分血清所识别的多肽相同,如1号、5号、6号、11号牛奶过敏患者血清所识别的表位均为1号多肽,有一部分牛奶过敏患者血清所识别的表位均不完全相同。根据多肽反应频率的不同,筛选出β-乳球蛋白最主要表位的氨基酸序列为AA76-95、AA1-20、AA106-125,主要表位的氨基酸序列为AA91-110、AA61-81、AA121-140,次要表位的氨基酸序列为AA 46-65、AA 31-50、AA 151-162。(2)β-乳球蛋白关键抗原表位串联体重组蛋白的表达及鉴定:通过DNAStar软件对理论上6种组合方式进行了分析,得到最佳组合方式,成功构建了大肠杆菌原核表达载体,IPTG诱导表达后,获得大小为36kDa左右的融合蛋白,并纯化得到单一组分的36kDa大小的重组蛋白,并经免疫印迹法、斑点印迹法鉴定其免疫活性,并利用重组蛋白作为检测抗原建立了一种活性氧传递均相发光免疫测定技术来检测血清sIgE。结论:本研究对牛奶主要过敏原β-乳球蛋白的抗原表位进行了解析,不同牛奶过敏患者血清所识别的抗原表位具有异质性。成功表达了β-乳球蛋白关键抗原表位串联体重组蛋白,并鉴定了关键抗原串联体重组蛋白具有良好的免疫学活性,在检测牛奶过敏疾病中有可能代替天然抗原,为新一代诊断试剂的制备奠定基础。
[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.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R446.6

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