利用免疫蛋白質體學分析幽門螺旋桿菌中與十二指腸潰瘍相關之抗原性蛋白質

Abstract

幽門螺旋桿菌為人類腸胃道病原菌，是一種螺旋狀的革蘭氏陰性菌，常感染胃部黏膜。全世界有超過50%的人口體內有此菌之存在，感染幽門螺旋桿菌的人，通常會伴隨慢性胃炎的症狀；在胃潰瘍和十二指腸潰瘍患者中，有很高的比率是幽門螺旋桿菌的帶原者。隨著慢性胃炎的持續發展，可能會導致胃腺癌或是胃部淋巴癌(MALT)的發生。1982年澳洲Marshall和Warren兩位醫師將此菌於人體外培養成功，而其具有1.67 Mb之基因體完整序列亦已於1997年發表，更引領相關研究之開展。 幽門螺旋桿菌本身會分泌許多的毒性因子，而幽門螺旋桿菌之外分泌性蛋白能與宿主細胞進行最直接的交互作用，引起細胞發炎反應或造成損傷；雖然已知兩個分泌性蛋白：VacA及CagA為幽門螺旋桿菌重要的毒性因子，在亞洲國家如日本、韓國及台灣，它們的表現與否和感染幽門螺旋桿菌造成的不同臨床結果並無相關，顯示可能有其他蛋白質扮演重要的角色。 由於十二指腸潰瘍與幽門螺旋桿菌的感染有高度相關，許多流行病學統計也指出，罹患十二指腸潰瘍病人得到胃癌的風險非常低，幾近於零；因此若比較十二指腸潰瘍與胃癌兩者間之差異性，應可發現幽門螺旋桿菌中與兩者相關的保護或致病因子。於是我們以液態培養幽門螺旋桿菌十二指腸潰瘍菌株，收取培養基後，使用二維電泳分離蛋白，再與十二指腸潰瘍病人的血清反應。以LC-MS/MS之質譜技術，分析免疫轉漬後具抗原性的蛋白質。 選擇十二指腸潰瘍病人之血清進行的二維電泳免疫轉漬實驗，發現大部分與十二指腸潰瘍相關之抗原性蛋白質多位於分子量40kDa ~ 100kDa。找到具抗原性之蛋白質包括Urease beta subunit、Chaperonin GroEL、Flagellin A、FusA-homolog (yihK)、Flagellar hook protein、hypothetical protein HP1037、Aconitate hydratase等，選殖並表現hypothetical protein HP1037，以十二指腸潰瘍及胃癌病人血清做篩檢後，發現其具有鑑別十二指腸潰瘍與胃癌之潛力。將來再進一步放大篩檢的血清數量，確認其免疫反應強弱是否具有疾病鑑別性，可配合其他幽門螺旋桿菌相關因子，一起應用在疾病診斷上。

Helicobacter pylori (H. pylori), as a human pathogen, is a spiral-shaped bacterium colonizing the gastric mucosa. More than 50% of the human population is infected by H. pylori. Infected people with H. pylori undergo a chronic gastric inflammation. Both gastric and duodenal ulcers are strongly related to H. pylori infection. Persistent gastric inflammation could increase the risk of developing gastric adenocarcinoma (GC) or mucosal-associated lymphoid tissue (MALT) lymphoma. H. pylori was first cultured from the stomach of the infected patient by Marshall and Warren in Australia in 1982. The whole genome sequence was completed in 1997 and soon applied to further research. H. pylori produces several virulence factors, among these factors are secretory proteins, which enable H. pylori to interact with host epithelium cells directly and raise inflammation and cell damage. Two well-known pathogenic proteins that are secreted are VacA and CagA. However, there is no association between VacA or CagA status and clinical outcome in Asian countries, such as Japan, Korea and Taiwan. This phenomenon may suggest that there is still another factor affecting different clinical outcomes. H. pylori infection is highly correlated to duodenal ulcer development, and it seems that DU patients have much lower risk for progressing to gastric cancer. We assume that there are DU or GC-related factors which have the ability to distinguish these clinical divergent events. We separated proteins from liquid-cultured H. pylori with two dimensional electrophoresis (2DE) and probed with DU patients’ sera. LC-MS/MS was then applied to analysis immuno-reactive proteins. In our result, several DU associated immuno-reactive proteins were found by 2-DE combined with immunoblot analysis. Most of them were located at molecular weight ranging from 40 to 100 kDa including Urease beta subunit、Chaperonin GroEL、Flagellin A、GTP-binding protein, fusA-homolog (yihK)、Flagellar hook protein、hypothetical protein HP1037、Aconitate hydratase. One of these antigens, hypothetical protein HP1037, was cloned and expressed. The immuno-reactivity of recombinant HP1037 was tested by using a number of DU and GC patients’ sera, and this protein may have the ability to distinguish DU from GC. With further experiments enlarging sample sizes and confirming this immunogenic property, this immuno-reactive protein may apply to diagnosis combined with other biomarkers.