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標題: | 利用大腸桿菌表現幽門螺旋桿菌之蛋白酶HtrA及其功能性分析 Functional analysis of the Helicobacter pylori protease HtrA expressed in E. coli |
作者: | Pei-Yu Li 黎培鈺 |
指導教授: | 周綠蘋(Lu-Ping Chow) |
關鍵字: | 幽門螺旋桿菌 蛋白酶,HtrA, Helicobacter pylori protease HtrA, |
出版年 : | 2006 |
學位: | 碩士 |
摘要: | 幽門螺旋桿菌是由Marshall和Warren兩位科學家所發現,它是一個格蘭氏陰性菌,通常會寄生在人體的胃表皮細胞。根據統計,全球有超過50%的人口都是幽門螺旋桿菌的帶原者。在患有十二指腸潰瘍(DU)的病患中,約有超過95%的人都是幽門螺旋桿菌的帶原者;而在胃潰瘍患者中,約有80-90%的人為幽門螺旋桿菌的帶原者。感染幽門螺旋桿菌的人,通常會伴隨慢性胃炎的症狀,慢性胃炎有可能會導致胃腺癌(GC)或是胃部淋巴癌(MALT)的發生。幽門螺旋桿菌本身會分泌許多的毒性因子,而這些毒性因子可能參與在幫助幽門螺旋桿菌在胃中的寄生或是能夠對宿主細胞造成傷害。
在先前的研究中,藉由利用酸性甘胺酸的方法萃取幽門螺旋桿菌之表面蛋白,結合二維電泳(2-DE)以及免疫染色法,我們發現一個幽門螺旋桿菌的蛋白-HtrA,它不論是對患有胃癌或是十二指腸病人的血清都有很高的抗原性。 HtrA本身是一個絲胺酸蛋白酶,它普遍存在於許多的原核生物中,甚至某些真核生物中也存在有HtrA。在本次的研究中,證實了HtrA是一種幽門螺旋桿菌的外分泌蛋白。為了探討HtrA本身的蛋白酶活性和幽門螺旋桿菌感染之致病性是否相關,我們針對HtrA的活性區,利用定點突變的方法來製造HtrA突變株。經由活性分析,我們發現HtrA的突變株已經完全喪失蛋白酶的活性。利用wtHtrA來處理人類周邊血液單核球細胞(PBMC),我們發現wtHtrA可以刺激PBMC釋放前發炎細胞激素,如IL-8、IL-6、IL-1β以及TNF-α,並且wtHtrA刺激PBMC釋放細胞激素是劑量相關的形式。此外,wtHtrA可以針對胃腺癌細胞AGS間的tight junction蛋白–occludin進行破壞;相對的,mtHtrA則不能破壞occludin。因此,HtrA似乎會參與在幽門螺旋桿菌感染所引發的發炎反應中,並且可能破壞胃表皮細胞間的連結,而且這樣的破壞是和它本身蛋白酶活性相關。 Helicobacter pylori(H. pylori) is first discovered by Marshall and Warren, as a gram-negative bacterium which colonized the gastric epithelium. According to the survey, more than 50% of the human population was infected by H. pylori. Among infected people with H. pylori, over 95% develop duodenal ulcers (DU) and 80-90% are affected by gastric ulcers. Infected persons with H. pylori undergo a chronic gastric inflammation with an increased risk of developing gastric adenocarcinoma (GC) or mucosal-associated lymphoid tissue(MALT) lymphoma. H. pylori generates several virulence factors. These virulence factors enable H. pylori to colonize under the gastric mucosa cells or make damage to the host epithelium cells. In the previous study, acid-glycine was used to extract surface proteins of H. pylori. Combining two-dimensional electrophoresis (2-DE) and immunoblotting, we found a H. pylori protein-HtrA, with high immuno-reactivity to the serum of GC and DU patients. HtrA is a serine protease found in many prokaryotes, and even some eukaryotes. In this study, HtrA was found to be secreted out of H. pylori. In order to investigate whether its serine protease property is involved in the pathogenesis induced by H. pylori infection, HtrA mutants (mtHtrA) were generated using site directed mutagenesis at its active sites and were confirmed the loss of protease activity by the zymogram experiment. In peripheral blood monocytes, wild type HtrA(wtHtrA) stimulated the production of IL-8, IL-6, IL-1β and TNF-α in a dose-dependent manner. Moreover, wtHtrA tended to disrupt occludin, one of the tight junction proteins in AGS cell whereas mtHtrA had no effects at all. Therefore, HtrA seems to be involved in the inflammatory response of H. pylori infection and disruption in paracellular junction of gastric epithelial cells by its protease activity. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/32437 |
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顯示於系所單位: | 生物化學暨分子生物學科研究所 |
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