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研究生:樊書傑
研究生(外文):Shu-Jie Fan
論文名稱:幽門螺旋桿菌之HP1492結構及功能探討
論文名稱(外文):Structural and functional studies of HP1492 from Helicobacter pylori
指導教授:程家維
指導教授(外文):Jye-Wei Cheng
學位類別:碩士
校院名稱:國立清華大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
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
論文頁數:54
中文關鍵詞:幽門螺旋桿菌
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幽門螺旋菌是一種微需氧的革蘭氏陰性桿菌,其具有4~6根鞭毛,使得細菌具有高度的移動能力。全世界大約有五成人口受到幽門螺旋菌的感染,一般認為幽門螺旋菌和胃潰瘍、十二指腸潰瘍、胃癌等消化道疾病的發生有關。為了解人類重大疾病之致病菌,我們選擇了幽門螺旋桿菌作為整個結構基因體計畫的主角,而本篇論文主要探討基因HP1492結構及功能性。
鐵硫蛋白在多種生物體內被發現,且扮演著重要的生理角色如:電子傳遞及代謝反應。雖然鐵硫蛋白在細胞功能及酵素反應中被廣泛的學習,但形成鐵硫中心的分子機制到目前還是不清楚。在我們的學習中,HP1492與鐵硫蛋白NifU 的C端有著相似的序列,其中包含了一段被認為是形成鐵硫中心重要的區段 ”C-X-X-C”。
為了瞭解HP1492在結構及功能上的特性,設計了一連串的實驗;從物理化學方面,由旋光儀得知HP1492有著很高的熱穩定性,Cm值也高達3.7M Urea,證明其結構上的穩定度。在運用定點突變法研究三個Cys,我們發現HP1492的三個Cys皆可能與Fe的鑑結上扮演重要的角色。最後在結構方面,雖說因雙體造成光譜上的複雜度,但藉由單一突變C79A樣品得到較穩定的HSQC,與未經突變樣品的光譜重疊後,其差異並不大,二級結構旋光光譜上也無太大改變,顯示出將可以此突變進行HP1492在結構方面相關的研究。
Helicobacter pylori is a micro-aerophilic Gram-negative bacillus. It has 4~6 flagella enables the bacterium to have the high motion ability. There are probably 50% population to be infected by the H. pylori all over the world. The infection of the H. pylori leads to serious disease such as duodenal or gastric ulcer, gastric adenocarcinoma, or mucosa-associated lymphoid tissue lymphoma. In order to realize the acute disease cause by the bacteria, we selected the H. pylori to be the target of structural genomics project. The topic of my research is about the structural and functional of HP1492.

Iron-sulfur proteins are distributed among various organisms and are known to play important physiological roles not only in electron transfer or metabolic reactions but also in gene regulation. Although cellular functions and enzymatic reactions of many iron-sulfur proteins have been extensively studied, molecular mechanisms involved in the assembly of the iron-sulfur cluster into the polypeptides still remain unclear. In our study, the HP1492 NifU homolog shares high sequence homology with the carboxyl-terminal domain and contains the conserved Cys-X-X-Cys motif of A. vinelandii NifU.

In order to understand the structure and the function of HP1492. We designed a succession of experiment. We used the Circular Dichroism to know the Tm value of HP1492 may reach as high as 82 ℃, the Cm value also reaches as high as 3.7M Urea. This result shows that HP1492 has high stability. We study three Cys of HP1492 by site-directed mutagenesis. We found that all of these three Cys play an important role in binding with Fe. Finally in the structural study, the high population of the homodimer make the structural study by NMR be more difficult. Site-directed mutagenesis of C79A reducing the formation of homodimer. The spectrums of HSQC and the stability detected by CD are similar to wild type HP1492. Thus site-directed mutagenesis of C79A may be used for the structural study.
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