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  • 學位論文

表現登革熱二型病毒NS2,NS4基因及探討四環黴素衍生物對登革熱病毒的抑制作用

Functional Expression of NS2 & NS4 of Dengue Virus 2 and Mechanism Study of Tetracycline-Derivatives as Dengue Virus Inhibitors

指導教授 : 楊昀良

摘要


登革熱目前已成為一個全球性健康的議題,主要分布在熱帶及亞熱帶地區。目前統計超過一百個國家曾經爆發過登革熱疫情並且大約有二十五億的人口受到登革熱病毒的威脅。因此目前針對登革熱疫苗的研發,治療和預防的方法也是一個迫切的議題。 在登革熱病毒產生蛋白質的過程,會先產生單一個蛋白質轉譯區,再切割成十個獨立的蛋白質。其中,NS2A、NS2B、NS4A及NS4B是較小的非結構型蛋白,具有疏水的特性。這四種蛋白質中,只有NS2B已知會參與蛋白切割反應,而NS2A、NS4A、及NS4B尚未被鑑定出確切的生化功能。為研究登革熱病毒此四蛋白質的結構及功能,本研究嘗試將四種蛋白質在哺乳類細胞中以加上EGFP (enhanced green fluorescence protein)的融合蛋白(fusion protein)形式表現。成功篩選出NS2A及NS2B穩定轉植細胞株後,溶斑試驗(plaque assay)的結果顯示:表現NS2A的穩定轉植細胞株經登革熱病毒感染後,相較於對照組會減少44%的溶斑數 (P <0.05)。 另外先前實驗室已經篩選出某些以四環結構為主體的四環黴素衍生物,其可與外膜蛋白上的β-OG pocket docking,也對登革熱二型病毒有初步的抑制效果。因此我將進一步利用溶斑試驗測試這些四環黴素衍生物針對登革熱病毒二型PL046病毒株及三型H87病毒株的抑制是否有strain specificity的效果。結果顯示,在溶斑試驗當中四環黴素衍生物對於登革熱病毒二型和三型的溶斑生成確實是有抑制的效果,其中又以doxycycline和chlortetracycline抑制的效果最為顯著。而進一步的比較發現,針對登革熱病毒二型的抑制能力比三型的抑制能力高。

並列摘要


Dengue viral infections have become a global health issue in tropical and subtropical regions. There were outbreaks reported in more than 100 countries and about 2.5 billion people lived under the threats. Therefore, the developments of vaccines, treatments, or prevention measures for dengue viral infections are a pressing issue. Dengue virus encodes 10 proteins in a single open reading frame. Among them, NS2A, NS2B, NS4A and NS4B are small non-structural proteins exhibiting hydrophobicity profiles. NS2B has been suggested to involve in protease activity while specific biological functions for NS2A, NS4A and NS4B have not been identified. With the interest to study the structure and function of these four proteins, I attempted to clone, express, and functional assay of these four genes. Their proteins were expressed as EGFP (enhanced green fluorescence protein) fusion proteins in mammalian cells. The stable cell lines of NS2A and NS2B were successfully selected and confirmed by Western blot analysis. The results showed that NS2A-EGFP effected a 44% reduction of plaque numbers compared to the negative controls, BHK-21 cells (P <0.05). Previously, our lab has identified certain tetracycline derivatives as potential inhibitors to Dengue virus, by docking into the β-OG pocket on the DV2 E protein. Hence, I set out to test various tetracycline compounds for their effect on dengue viral propagation of DV2 PL046 strain and DV3 H87 strain by plaque formation assay. All the tetracycline derivatives tested inhibited the plaque formations on DV2 and DV3 in cell culture systems, especially of doxycycline and chlortetracycline. And in comparison, the inhibitory effect on DV2 is better than that on DV3.

參考文獻


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