【摘要】：背景： 貓過敏原是常見的室內(nèi)吸入性過敏原，在世界范圍內(nèi)普遍參與IgE介導的過敏反應。Fel d1作為貓過敏原中最主要的致敏原，目前已成為開發(fā)新的變應原特異性免疫治療（Allergen-specific immunotherapy，ASIT）的理想蛋白分子。細胞因子在過敏性炎癥反應中起著非常重要的調節(jié)作用。其中，IL-10是一種具有多種生物學活性的免疫抑制因子，對機體產(chǎn)生保護作用。本研究在實驗室已經(jīng)構建好的Fel d1-IL-10（FIL）重組嵌合蛋白的基礎上，為了降低重組蛋白的過敏原性，對該嵌合基因中Fel d1MHC-II類的抗原性進行綜合分析，確定抗原改造的關鍵位點，通過基因工程定點突變技術對重組貓過敏原氨基酸序列中抗原值高的位點進行改造，將改造后的基因序列導入載體，轉化入大腸桿菌中進行誘導表達。免疫學鑒定后經(jīng)可溶性分析已得到較純的目的蛋白，進而為下一步目標蛋白功能試驗打下堅實的基礎。 目的： 運用生物信息學軟件分析過敏原Fel d1MHC-II類抗原性表位結合力，在保證蛋白質三級結構不變的基礎上，對其抗原性進行有目的的改造，得到抗原性弱化的低過敏原制劑，并期望通過IL-10的免疫調節(jié)作用，降低由Fel d1產(chǎn)生的免疫應答，誘導免疫耐受，從而獲得易于標準化和安全高效的貓過敏原制劑，為進一步研發(fā)變應原疫苗應用于臨床治療提供理論支持和物質保證。 方法： 1.使用在線軟件NetMHCII2.2Server分析貓主要過敏原Fel d1氨基酸序列的抗原性表位結合力，確定需要改造的高結合力位點即高值位點； 2.在保證改造前后蛋白質三級結構無改變的基礎上，遵循抗原改造的原則，對需要改造的位點使用極性小、側支少以及無芳香環(huán)的氨基酸取代該位點上極性大、側支多以及有芳香環(huán)的氨基酸，，得到抗原性低的序列； 3.使用基因工程的方法對選定的高值位點進行定點突變，誘導表達和純化目的蛋白； 4.對各突變重組蛋白進行免疫學鑒定。結果： 1.預測得到貓主要過敏原Fel d1上二個高值MHC-II抗原表位位點； 2.在此兩個抗原表位的基礎上，構建了三個突變重組蛋白。 結論： 對Fel d1-IL-10嵌合基因中過敏原抗原性進行了分析，預測出二個高值MHC-II抗原表位位點，并針對這兩個位點進行定點突變，構建了三個重組蛋白突變體，為下一步重組蛋白突變體的功能檢測和驗證打下了堅實的基礎。
[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.
【學位授予單位】：廣州醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R392

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