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    題名: TLR5競爭物之類鼻疽鞭毛修飾DNA質體的免疫機制研究(III)
    Mechanisms of DNA Immunization Using a Modified Plasmid Encoding the TLR5 Agonist-Burkholderia Pseudomallei flagellin(III)
    Authors: 陳亞雷;陳垚生;高志明
    Ya-Lei Chen
    貢獻者: 生物科技系
    Date: 2008-08
    Issue Date: 2010-12-31 16:41:58 (UTC+8)
    Abstract: 基礎研究, 研究期間 9708~ 9807, 研究經費 1042 千元
    Burkholderia pseudomallei 之flagellin (fliC)具有modulin 性質,可誘發 macrophage 釋放inflammation cytokines,其作用機制可能是利用N-terminal conserved regions 具有XRXR motifs 的氨基酸序列(稱之為TLR5 agonist),與target cell 表面的TLR5 結合,進而啟動了inflammation response。因此,構築TLR5 agonist 與interest gene 的hybrids 於plasmid DNA vaccine 上,期望藉由TLR5 調節之發炎反應,大幅提高plasmid DNA vaccination 的免疫抗原性,以解決現今 DNA 疫苗發展上的限制。為探索TLR5 agonist 的DNA vaccine 機制,我們構築人工合成之TLR5 agonist 與GroEL (interest gene)的hybrid,同時亦構築fliC (TLR5 ligand)與GroEL 的hybrid 為陽性比較組。FliD 蛋白具有類似fliC 的結構,有N-、C-terminal conserved regions,但缺乏TLR5 ligand (XRXR motifs)。因此,亦構築fliD-GroEL hybrid 為陰性比較組,資以證明TLR5 agonist 在DNA vaccination 時的佐劑功效。我們預期先於試管實驗中,證實這些hybrid gene 產物保有TLR5 agonist 時,可誘發macrophage 發生inflammation response,同時不會受哺乳類細胞蛋白質表現時,所發生的糖基化影響。我們並進一步利用動物實驗,證明TLR5 agonist- GroEL hybrid gene 注射後,確實可誘發dendric cell 成熟,表現B7 分子。我們亦對疫苗注射後,進行免疫性分析,評估所誘發之humoral 與cellular immune response 的能力。既然認為DNA vaccine 是誘發Th-1 immune response 的重要疫苗策略,因此,我們特別評估經TLR5 agonist-GroEL hybrid 進行質體免疫後,所誘發的Th-1 related cytokines 是否能激活macrophage,產生有效的opsonic killing 效果,以及質體免疫後,所活化的dendrtic cell 是否可有效的活化Tc cell,發生有效率的 cytotoxic effect。最後,我們將以Pseudomonas aeruginosa 感染免疫TLR5 agonist-GroEL hybrid 的實驗動物,檢視DNA vaccine 的保護成效。現今DNA 免疫策略,常受限於不易產生高免疫抗原性的限制,利用TLR5 agonist 修飾的DNA 疫苗,將有可能解決此項限制。藉由本計劃之實施,將可了解TLR5 agonist 在DNA 免疫的機制,並可進一步應用在各種DNA 疫苗的發展上。
    The flagellin of Burkholderia pseudomallei flagellin, acts as modulin, can induce the release of inflammatory cytokines by macrophage. This response is initially through the recognition between XRXR motifs (called TLR5 agonist) of flagellin and TLR5 on the surface of target cells and furthermore affects the acquired immune response. Thus, the hybrid genes in plasmid DNA were constructed by linking of TLR5 agonist and interest gene and then immunized into animals. The highly immunogenecity in animals will be generated if TLR5 agonist can induce inflammatory response in vivo. The hybrid genes including TLR5 agonist-GroEL, fliC-GroEL and fliD-GroEL will be constructed for DNA vaccination. Amongst of them, the fliC, a well know TLR5 ligand, used as positive controls and fliD, contains N- and C-terminal conserved regions to fliC but without XRXR motifs (TLR5 agonist), used as negative controls. At first, we demonstrate that hybrid gene product with TLR5 agonist even have a post-translational modification (glycosylation) can induces inflammatory response in vitro. Subsequent to Balb/c mice immunized with hybrid gene plasmids, the expression of B7 molecules on dendric cell of the animals can be induced. Also, the humoural or cellular immune response was evaluated at indicated time in the mice subsequent to commencement of vaccination. In particularly, the resident macrophage can be activated though the interaction with Th1-related cytokines from spleen cells released in immunized mice. The opsonic killing effect by activated macrophage will be indeed increased. The cytotoxicity by Tc cells is also evaluated when the Tc cell have been interacted with dendric cells, isolating from spleen of immunized mice. Finally, the Balb/c mice immunized with this modified plasmid by TLR5 agonist are infected with Pseudomonas aeruginosa to evaluate the protection against this pathogen eventually. In further study, the DNA vaccine with TLR5 agonist modification will apply to prophylaxis for a number of pathogens.
    關聯: 行政院國家科學委員會計畫 / 計畫編號 NSC95-2320-B017-002-MY3
    Appears in Collections:[生物科技學系] 研究計畫
    [生科系] 陳亞雷

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