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研究生:黃則豪
研究生(外文):Tse-Hao Huang
論文名稱:重組家塵蹣過敏原Derp7之純化、特性分析與晶體結構測定
論文名稱(外文):Purification, Characterization and Structural Determination of the Recombinant Group 7 Allergen from Dermatophagoides pteronyssinus
指導教授:廖淑惠廖淑惠引用關係
指導教授(外文):Shwu-Huey Liaw
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生物化學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
中文關鍵詞:家塵□過敏原
外文關鍵詞:House dust miteAllergen
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家塵□已被認為是主要的室內過敏原,是引起氣喘、過敏性鼻炎、異位性皮膚炎...等過敏性疾病的重要因子。在全球多種常見的□種中,Dermatophagoides pteronyssinus 是台灣最猖獗的種類。家塵□第七組過敏原(Der p 7)基因是由沈弘德博士所選殖,含215個胺基酸:N端的17個胺基酸之導引片段(leader peptide)與198個胺基酸之成熟蛋白,其蛋白功能未知。IgE的結合試驗與皮膚測試顯示此蛋白質會辨認約50%塵□過敏病人血液中的IgE,且具有與主要過敏原Der p 1類似之刺激細胞素產生能力。目前為止,尚未發現Der p 7之同源性(homology)蛋白,因此推測Der p 7可能為一個新的立體結構(novel structural fold)。
為探討Der p 7的過敏反應機制、IgE可能的結合位置與此新穎立體結構,在此論文我先純化大腸桿菌所表現的重組Der p 7,圓二色光譜(circular dichroism)顯示rDer p 7為一□-helical蛋白。分析型超高速離心實驗(analytical ultracentrifugation)顯示,rDer p 7在溶液中主要是以單體(monomer)存在,少部分則以三聚體(trimer)和六聚體(hexamer)存在。另外也培養出Der p 7蛋白晶體,其結晶溶液為20 % polyethylene glycol 4000,20 mM ammonium acetate and 100 mM sodium citrate (pH 5.6),溫度為15℃,一旦有晶體生成,便將晶體培養皿移至4℃冰箱,需存放一週以上,使用台北榮總X光繞射儀分析,晶體繞射能力為4 □左右,因訊號太弱而無法進行分析。因此須使用光源很強的同步輻射,如日本SPring-8 BL-41XU光束線,進行繞射數據收集與分析。其X光繞射點的解析度為3.2 □,空間群屬於P212121,晶胞大小為a = 53.4 □, b = 79.8 □, c = 366.6 □。由於晶格c軸很長,所以在繞射數據收集時,晶體c軸須與goniometer轉軸平行,以避免繞射點重疊。已收集到三組Se-MAD資料,self-rotation function分析有6-fold的非晶體對稱軸,Se-MAD初步分析與晶格排列顯示,含有2-和3-fold 非晶體對稱軸,其2-fold軸略平行於x軸,而3-fold軸則平行z軸。因此經由Matthew’s coefficient顯示一個asymmetry unit中應含有六個蛋白分子。另外,由3D-PSSM預測 Der p 7結構也許會類似人類的matrix protein p32。綜合以上分析推論,同一分子內,N端與C端的helices會形成coiled coils,希望在這些資訊的輔助之下,能找到2-和3-fold NCS (non-crystallographic symmetry)之位置,進而運用6-fold average 來加速晶體結構之測定。
Allergic diseases such as atopic dermatitis, rhinitis and asthma afflict more than 20% of the world population. House dust mites have been regarded as one of the most important sources of indoor allergens. The two most important clinical mite species are Dermatophagoides pteronyssinus and Dermatophagoides farinae. The group 7 allergen from D. pteronyssinus (Der p 7) consists of a 17-residue leader peptide and a 198-residue mature protein. This allergen has been shown to react with about 50% of allergic sera. Our sequence analysis suggests that Der p 7 does not share significant sequence homology to any known proteins and hence may contain a novel structural fold. To gain structural insights into the allergenicity mechanism, we have obtained the first Der p 7 crystals and attempt to solve the phase problem using the selenomethionyl multiwavelength anomalous dispersion (Se-MAD) method.
Circular dichroism measurements suggest that Der p 7 is a helical protein. Analytical ultracentrifugation analysis reveals that Der p 7 exists in solution mainly as a monomer with trace amounts of trimer and hexamer. The Der p 7 crystals have been grown at 15 oC in 20% polyethylene glycol 4000, 20 mM ammonium acetate and 100 mM sodium citrate (pH 5.6). The crystals belong to the P212121 space group with unit cell dimensions of a = 53.4 □, b = 79.8 □, c = 366.6 □. The crystals diffract x-rays beyond 3.2 □ resolution. A self-rotation search revealed a six-fold point symmetry and thus suggested six monomers in each asymmetric unit.
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