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研究生:曾凱莉
研究生(外文):Kai-li Tseng
論文名稱:胃幽門螺旋桿菌細胞毒素相關基因A的基因選殖與在哺乳動物細胞中的表達
論文名稱(外文):Molecular Cloning of Helicobacter pylori Cytotoxin-Associated Gene A and Its Expression in Mammalian Cells
指導教授:傅化文傅化文引用關係
指導教授(外文):Hua-wen Fu
學位類別:碩士
校院名稱:國立清華大學
系所名稱:分子與細胞生物研究所
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:英文
論文頁數:54
中文關鍵詞:胃幽門螺旋桿菌細胞毒素相關基因A第四類分泌系統蜂鳥形表型
外文關鍵詞:Helicobacter pyloricagAtype IV secretion systemhummingbird phenotype
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胃幽門螺旋桿菌已知是引起胃炎、消化性潰瘍,甚至是胃癌的主要原因之一。它感染在人類的胃部,且現今在世界人口中的感染率已達五成。在胃幽門桿菌所產生的毒性因子中,其中的「細胞毒性相關基因A」 (cytotoxin-associated gene A; cagA) 所產生的毒素,被認為在引起上述較嚴重的胃部疾病中扮演重要角色。然而,其之所以致病的詳細分子機制卻仍有待研究。過去已知在具cagA基因的胃幽門桿菌菌株的感染中,CagA會被經由細菌的第四類分泌系統 (type IV secretion system) 直接注入到宿主表皮細胞內。進入到細胞中的CagA會被宿主細胞的Src家族磷酸化酵素在CagA上的絡胺酸(tyrosine)處磷酸化,而被磷酸化的絡胺酸則是在麩胺酸(glutamic acid; E)-脯胺酸(proline; P)-異白胺酸(isoleucine; I)-絡胺酸(tyrosine; Y)-丙胺酸(alanine; A)的五胺基酸序列(EPIYA)中。當CagA被磷酸化後,會引起宿主細胞其細胞骨架之重組,產生所謂「蜂鳥形表型」(hummingbird phenotype),亦即細胞伸展延長,類似蜂鳥嘴巴形狀的外觀。另一方面,不同菌株的CagA蛋白質在羧基端(C-terminal)部分的胺基酸序列具有相當的差異,而這些差異被認為是不同菌株之所以造成不同疾病的原因之一。另外,各菌株的分佈亦存在著地理區域上的差異,這些地理分佈上的差異也可能與不同地區胃癌盛行率不同有關。為了研究CagA對哺乳動物細胞的影響,在此論文中,我建構了以pEGFP-N1質體為基礎的cagA基因哺乳動物細胞表現載體並於哺乳動物細胞株中表達。我發現在人類胃黏膜上皮細胞株(AGS cells)中有些細胞確實產生延展及分散的型態,然而,這些載體所表現出的CagA蛋白質量卻很低,幾乎無法偵測到,從這些結果,我推測此系統的載體對於在哺乳動物細胞中表達cagA基因並不合適。此外,我也做了26695與NCTC11637兩菌株的胺基酸序列比對;亦比對了五株台灣菌株、另一東亞菌株(F32)及26695菌株在羧基端部分的胺基酸序列。從這些序列比對的結果中,可以推測不同的序列很可能導致不同的CagA活性,進而造成胃幽門螺旋桿菌感染時不同的結果。這些序列上的差異,對於臨床上疾病的發展、預後,亦可作為一參考的指標。
Helicobacter pylori (H. pylori) is the major causative agent of gastritis, peptic ulcer diseases and even gastric cancer. This pathogen colonizes the human stomachs of at least half of the world’s population. Cytotoxin-associated gene A (cagA), one of the virulence factors generated by H. pylori, is considered to play an important role in the pathogenesis of these diseases. However, the molecular mechanism under the pathogenesis is still unclear and needs to be identified. It has been known that during cagA+-H. pylori infection, CagA is translocated into host epithelial cells via type IV secretion system. Translocated CagA is tyrosine phosphorylated on specific EPIYA sequence repeats by cellular Src family tyrosine kinases. Phosphorylation of CagA induces rearrangements of host-cell actin-cytoskeleton and cell scattering, resulting in the so-called “hummingbird phenotype”. On the other hand, CagA from different strains varies in sequences in the C-terminal region. Strain-specific genetic diversity of CagA has been proposed to be involved in the ability of different H. pylori strains to cause different diseases. There are also indications of significant geographic differences among strains. In order to study the effect of CagA in mammalian cells, in this study, I constructed pEGFP-N1 based cagA-expression vectors and expressed cagA in mammalian cells. I found that in AGS cells transfected with these cagA-expression vectors, some cells exhibited elongated and scattering phenotypes. However, the expression level of CagA was low. The result indicated that pEGFP-N1 based vectors might not be suitable for ectopically expressing cagA. In addition, I also performed total amino-acid sequence alignment of two H. pylori strains (26695 and NCTC11637) and sequence alignment of C-terminal region of five Taiwan strains with those of another East Asian strain (F32) and strain 26695. The result suggested that the presence of particular sequences might probably correlate with different activities of CagA. These sequences may serve as indications of different disease outcomes of different H. pylori infection.
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