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    Please use this identifier to cite or link to this item: http://ir.lib.ncu.edu.tw/handle/987654321/49490


    Title: 構建大腸桿菌K1蛋白質體晶片並用來發展研究人類細胞與病原菌蛋白質體交互作用之新型分析方法;Construction of an Escherichia Coli K1 Proteome Chip and Its Application on the Novel Assay Development for the Interaction Study between Human Cells and Pathogen Proteome
    Authors: 陳健生;鄧景浩;阮雪芬
    Contributors: 系統生物與生物資訊研究所
    Keywords: 大腸桿菌蛋白質體晶片;高通量浸泡式基因轉殖;人腦微血管內皮細胞;血腦障壁;研究領域:醫學技術
    Date: 2011-08-01
    Issue Date: 2012-01-17 18:58:34 (UTC+8)
    Publisher: 行政院國家科學委員會
    Abstract: 蛋白質體晶片是一種新型的高通量技術,為有效率研究蛋白質體學的工具,它非常適合用來進行大規模篩選蛋白質,已被應用在各式各樣的生化及生物的研究上,其最大的特點之一就是可以系統化的進行研究。我們實驗室已經具備各種蛋白質晶片,包含了人類、酵母菌、大腸桿菌K12蛋白質體晶片以及多樣的抗體晶片。腦中樞神經系統的感染一直是造成高致病性與高致死率的重要疾病之一。細菌性腦膜炎是一種非常常見且普遍的疾病且也是腦中樞神經系統疾病的其中一個成因,在所有的微生物中,只有非常少的一部份有能力去入侵人類的腦部。大腸桿菌是K1一種革蘭氏陰性菌,並且是造成腦膜炎的主要病菌。目前已經有許多的研究是針對病原菌如何穿越血腦障壁的機制進行研究,血腦障壁主要由一群排列緊密的人腦微血管內皮細胞組成並且分隔了腦與血管,負責保護人腦免於一些充斥在血流中的微生物以及毒物的侵襲。因此,瞭解人腦微血管內皮細胞與大腸桿菌K1間的交互作用對於瞭解細菌性腦膜炎是很重要的課題,然而現有的知識關於致病菌如何入侵腦部仍然是不完整的。我們實驗室在先前的研究已經運用各式各樣的蛋白質晶片去進行研究,包含K12蛋白質體晶片及抗體晶片。因此,我們希望利用高通量浸泡式基因轉殖這一個適合做大規模基因轉殖的技術去建構出大腸桿菌K1的特殊的選殖資料庫,再利用此基因庫轉移載體進入適合表現蛋白質的細胞株去進行高通量蛋白質的表現及純化,並且做出大腸桿菌K1蛋白質體晶片。為了測試細胞與蛋白質晶片的直接偵測是否可行,我們先利用了大腸桿菌K12進行預實驗,發現此一方式是可行的,但是並不完美。因此,我們在這個計劃中將發展最佳化細胞與蛋白質晶片的實驗條件並且增加實驗的重覆性與穩定性,用此新穎的平台來找出更多與大腸桿菌K1入侵人類腦部有關的致病蛋白質。 Protein microarray recently emerged as a useful and powerful tool for high-throughput protein screening studies, has been applied to many kinds of investigations in biological and biochemistry researches. Various types of proteome chips have been fabricated and used to conduct experiments. One of the most unique points of proteome chips is the ability of systematic study. Central nervous system (CNS) infections continue to be a crucial factor for morbidity and mortality throughout the world. Bacterial meningitis is one of the most common and well known types that cause CNS infections, among all the microorganisms, only a small portion of them are capable to infect human brains. Escherichia coli K1 is a major gram-negative organism causing meningitis; many studies have focused on how this pathogen penetrating the blood brain barrier (BBB), which separates circulating blood from cerebrospinal fluid (CSF) in the CNS. BBB is formed mainly by human brain micro-vascular endothelial cells (HBMEC) and protects the brain from any organisms and toxins circulating in the blood. The interaction between HBMEC and microorganisms is crucial for people to develop new strategies to prevent meningitis; however, it is still unclear how pathogens that penetrate HBMEC. In our lab, we already have the experience to deal with many kinds of protein microarrays, including yeast proteome chip, E. coli K12 proteome chip and antibody microarray. Also, this high-throughput and convenient methodology has been applied to versatile researches. Hence, we want to use infusion cloning method which is compatible for high-throughput cloning steps to construct about 480 E. coli K1 specific clone library. Also, we will transform these vectors into expression cells and to express and purify the proteins in a high-throughput manner. After that, we will fabricate E. coli K1 proteome chips and use this tool to study the cell-protein interactions.We have done pilot tests to use E. coli K12 proteome chip interact with HBMEC and showed promising results. This is the first time using human cells to interact directly with genome-wide bacteria proteins. This method still has room to be improved; hence, in this project we will construct E. coli K1 specific gene library and fabricate the E. coli K1 proteome chip, finally, we will build a robust model to investigate human cells with protein chips and use this methodology to reveal more virulent factors of E. coli K1 研究期間:10008 ~ 10107
    Relation: 財團法人國家實驗研究院科技政策研究與資訊中心
    Appears in Collections:[系統生物與生物資訊研究所] 研究計畫

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