猫过敏原重组蛋白突变体的构建及免疫学鉴定

发布时间：2018-10-18 10:21

【摘要】：背景：猫过敏原是常见的室内吸入性过敏原，在世界范围内普遍参与IgE介导的过敏反应。Fel d1作为猫过敏原中最主要的致敏原，目前已成为开发新的变应原特异性免疫治疗（Allergen-specific immunotherapy，ASIT）的理想蛋白分子。细胞因子在过敏性炎症反应中起着非常重要的调节作用。其中，IL-10是一种具有多种生物学活性的免疫抑制因子，对机体产生保护作用。本研究在实验室已经构建好的Fel d1-IL-10（FIL）重组嵌合蛋白的基础上，为了降低重组蛋白的过敏原性，对该嵌合基因中Fel d1MHC-II类的抗原性进行综合分析，确定抗原改造的关键位点，通过基因工程定点突变技术对重组猫过敏原氨基酸序列中抗原值高的位点进行改造，将改造后的基因序列导入载体，转化入大肠杆菌中进行诱导表达。免疫学鉴定后经可溶性分析已得到较纯的目的蛋白，进而为下一步目标蛋白功能试验打下坚实的基础。目的：运用生物信息学软件分析过敏原Fel d1MHC-II类抗原性表位结合力，在保证蛋白质三级结构不变的基础上，对其抗原性进行有目的的改造，得到抗原性弱化的低过敏原制剂，并期望通过IL-10的免疫调节作用，降低由Fel d1产生的免疫应答，诱导免疫耐受，从而获得易于标准化和安全高效的猫过敏原制剂，为进一步研发变应原疫苗应用于临床治疗提供理论支持和物质保证。方法： 1.使用在线软件NetMHCII2.2Server分析猫主要过敏原Fel d1氨基酸序列的抗原性表位结合力，确定需要改造的高结合力位点即高值位点； 2.在保证改造前后蛋白质三级结构无改变的基础上，遵循抗原改造的原则，对需要改造的位点使用极性小、侧支少以及无芳香环的氨基酸取代该位点上极性大、侧支多以及有芳香环的氨基酸，，得到抗原性低的序列； 3.使用基因工程的方法对选定的高值位点进行定点突变，诱导表达和纯化目的蛋白； 4.对各突变重组蛋白进行免疫学鉴定。结果： 1.预测得到猫主要过敏原Fel d1上二个高值MHC-II抗原表位位点； 2.在此两个抗原表位的基础上，构建了三个突变重组蛋白。结论：对Fel d1-IL-10嵌合基因中过敏原抗原性进行了分析，预测出二个高值MHC-II抗原表位位点，并针对这两个位点进行定点突变，构建了三个重组蛋白突变体，为下一步重组蛋白突变体的功能检测和验证打下了坚实的基础。
[Abstract]:Background: cat allergen is a common indoor inhalational allergen and is widely involved in IgE mediated allergic reactions worldwide. Fel D1 is the most important allergen in cat allergen. At present, it has become an ideal protein molecule for the development of new allergen specific immunotherapy (Allergen-specific immunotherapy,ASIT). Cytokines play an important role in the regulation of allergic inflammation. Among them, IL-10 is a kind of immunosuppressive factor with many biological activities, which has protective effect on organism. In order to reduce the allergenicity of the recombinant Fel d1-IL-10 (FIL) chimeric protein, the antigenicity of Fel d1MHC-II class in the chimeric gene was analyzed synthetically to determine the key site of antigen modification on the basis of the Fel d1-IL-10 (FIL) recombinant chimeric protein that had been constructed in the laboratory. The site with high antigen value in the amino acid sequence of the recombinant cat allergen was modified by genetic engineering site-directed mutation technique. The modified gene sequence was introduced into the vector and transformed into Escherichia coli for induction and expression. After immunological identification, a relatively pure target protein was obtained by soluble analysis, which laid a solid foundation for the further functional test of the target protein. Objective: to analyze the antigenic epitope binding ability of allergen Fel d1MHC-II by bioinformatics software, and to modify the antigenicity of antigenicity on the basis of keeping the tertiary structure of protein unchanged. A low allergen preparation with attenuated antigenicity was obtained, and it was expected to reduce the immune response produced by Fel D1 and induce immune tolerance through the immunomodulatory effect of IL-10, so as to obtain a cat allergen preparation which is easy to standardize and safe and efficient. It provides theoretical support and material guarantee for further research and development of allergen vaccine in clinical treatment. Methods: 1. Using online software NetMHCII2.2Server to analyze the antigenic epitope binding ability of Fel D1 amino acid sequence of main allergen in cats, and to determine the high binding site that needs to be modified, that is, high value site; 2. On the basis of ensuring that the tertiary structure of protein remains unchanged before and after modification, and following the principle of antigen modification, the polarity of the site in need of modification is small, the amino acid with less lateral branches and no aromatic ring is used to replace the polarity of the site. Low antigenicity sequence of amino acids with many collateral and aromatic rings was obtained. The target protein was expressed and purified by site-directed mutation at selected high value sites by genetic engineering. 4. The mutated recombinant proteins were identified by immunology. Results: 1. Two high value MHC-II epitopes on the main allergen Fel D1 were predicted. 2. On the basis of these two epitopes, three mutant recombinant proteins were constructed. Conclusion: the allergen antigenicity of Fel d1-IL-10 chimeric gene was analyzed, two epitopes of high value MHC-II antigen were predicted, and three recombinant protein mutants were constructed by site-directed mutagenesis. It lays a solid foundation for function detection and verification of recombinant protein mutants.
【学位授予单位】：广州医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392

【参考文献】