胃幽門螺旋桿菌嗜中性白血球激活蛋白之異源表現與功能分析

Heterologous Expression and Functional Analysis of Helicobacter pylori Neutrophil-Activating Protein

王仲安(Chung-An Wang)

指導教授 ： 傅化文

國立清華大學/生命科學院/分子與細胞生物研究所/碩士(2008年)

摘要

Helicobacter pylori (H. pylori) is the major pathogen which causes gastritis, peptic ulcer disease, and gastric cancer. H. pylori neutrophil-activating protein (HP-NAP) is an important virulence factor of H. pylori. Several studies showed that HP-NAP has the ability to activate innate and adaptive immune cells. In order to obtain sufficient HP-NAP, Escherichia coli (E. coli) and Bacillus subtilis (B. subtilis) were used to express recombinant HP-NAP. However, endotoxin in E. coli may affect HP-NAP-induced immune responses. The construction of B. subtilis expression system is also not well-known. Therefore, in the first part of this thesis, an efficient method was established to purify recombinant HP-NAP with low amounts of residual endotoxin from E. coli expression system. This purified recombinant HP-NAP was able to stimulate neutrophils to produce reactive oxygen species (ROS) and secrete myeloperoxidase (MPO). In the second part of this thesis, a B. subtilis expression plasmid containing nap gene was constructed and introduced into B. subtilis. Recombinant HP-NAP was expressed as a soluble and multimeric protein in B. subtilis. Taken together, these results indicate that E. coli-expressed recombinant HP-NAP has immunological activity to stimulate human neutrophils and that this newly established B. subtilis system can successfully express recombinant HP-NAP.

關鍵字

胃幽門螺旋桿菌 ； 胃幽門螺旋桿菌嗜中性白血球激活蛋白 ； 內毒素 ； 枯草桿菌

並列摘要

胃幽門螺旋桿菌是引發胃炎、消化性潰瘍，以及胃癌的主要致病菌。而胃幽門螺旋桿菌嗜中性白血球激活蛋白是胃幽門螺旋桿菌的重要致病因子。許多研究指出胃幽門螺旋桿菌嗜中性白血球激活蛋白具有活化先天性與後天性免疫細胞的能力。為了取得足夠的胃幽門螺旋桿菌嗜中性白血球激活蛋白，大腸桿菌與枯草桿菌已被用來表現重組胃幽門螺旋桿菌嗜中性白血球激活蛋白。然而，大腸桿菌中的內毒素可能會干擾胃幽門螺旋桿菌嗜中性白血球激活蛋白所誘發的免疫反應。關於枯草桿菌表現系統的建立也不甚清楚。因此，在本篇論文的第一部分裡，已建立一個有效的方法，藉此從大腸桿菌表現系統中純化出低內毒素殘留的重組胃幽門螺旋桿菌嗜中性白血球激活蛋白。此純化重組蛋白能夠刺激嗜中性白血球產生活性氧化物並且分泌骨髓過氧化酶。在本篇論文的第二部分裡，帶有胃幽門螺旋桿菌嗜中性白血球激活蛋白基因的枯草桿菌表現載體已被建構並送入枯草桿菌中。重組胃幽門螺旋桿菌嗜中性白血球激活蛋白能夠以可溶性與多聚體的形式表現於枯草桿菌之中。綜合上述，這些結果顯示大腸桿菌所表現的重組胃幽門螺旋桿菌嗜中性白血球激活蛋白具有活化人類嗜中性白血球的免疫活性，也證實了新建立的枯草桿菌系統可以成功地表現重組胃幽門螺旋桿菌嗜中性白血球激活蛋白。

並列關鍵字

Helicobacter pylori ； Helicobacter pylori neutrophil-activating protein ； endotoxin ； Bacillus subtilis

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