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  • 學位論文

腸病毒71型疫苗的開發:類病毒顆粒穩定性之探討

Development of EV 71 Vaccine:Studies on the Stability of Virus-like Particles

指導教授 : 胡育誠
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摘要


先前本實驗室已經證實利用桿狀病毒/昆蟲細胞表現系統所生產出的類病毒顆粒(virus-like particles,VLP)能有效誘發體內的免疫反應,且對於多種不同株的腸病毒71型皆有良好的中和效果,顯示類病毒顆粒確實有作為腸病毒71型疫苗的潛力。在本研究中,首先我們建立類病毒顆粒穩定性檢驗分析平台,同時進行類病毒顆粒的穩定性分析,並探討能增加其穩定性的最適化條件。在第一階段的研究中,我們利用粒徑分析儀及穿透式電子顯微鏡測量與觀察類病毒顆粒的動態特性(hydrodynamic properties)。首先,我們探討不同濃度的NaCl溶液、不同種類的緩衝溶液及穩定劑對於類病毒顆粒在4C環境下穩定性之影響,以粒徑分析儀分析類病毒顆粒的粒徑大小是否會隨著保存時間增加而變化,做為穩定性的判斷依據。而在第二階段的研究,我們採用酵素連結免疫吸附分析法分析類病毒顆粒的in-vitro antigenicity變化,評估在不同的保存條件下,類病毒顆粒隨著時間增加是否仍保持其活性,具有誘發免疫反應的能力。實驗結果顯示,類病毒顆粒的保存濃度會影響其穩定性,類病毒顆粒濃度越高,其穩定性越好,而類病毒顆粒在4C的保存環境中,保存後第5個月後仍具有極大的穩定性,添加穩定劑對於增加類病毒顆粒穩定性並無顯著效果。此外,類病毒顆粒保存於不同的緩衝溶液中也仍具有高度的穩定性,因此在生產、純化及最後保存類病毒顆粒的製程中,我們所使用的緩衝溶液並不會影響類病毒顆粒的穩定性,進而造成生產產率上的損失以及影響純化結果。因此,保存於4C及sodium phosphate (pH 6.5)溶液中是一個相當適合類病毒顆粒的環境,使得類病毒顆粒能維持高度的穩定性。綜合上述結果,本研究證實了在類病毒顆粒各個不同階段的的製程中,類病毒顆粒仍呈現穩定的狀態,也證實類病毒顆粒在4C之保存環境下非常穩定,不需額外添加穩定劑以增加其穩定性。

並列摘要


Enterovirus 71 (EV71) is an etiologic agent responsible for seasonal epidemics of hand-foot-and-mouth disease and causes outbreaks with significant mortality in child under 5 years of age, but there is no effective vaccine for EV71. Our group has previously demonstrated that EV71 virus-like particles (VLP) produced from the baculovirus/insect cell system induce high antibody titers in animal models and protect animals from virus infection, suggesting that VLPs are a good EV71 vaccine candidate. In this study, we aimed to investigate and improve the stability of EV71 VLPs. We first established the characterization methods including Dynamic Light Scattering (DLS), Transmission Electron Microscopy (TEM) and Enzyme-Linked Immunosorbent Assay (ELISA) to evaluate the VLPs stability and further examined the parameters that influence the VLPs stability, including buffer, temperature and stabilizer. The data demonstrated that the shape, size and immunogenicity of VLPs remained stable when stored in the buffers used in the VLPs production and purification processes. The VLPs also remain stabled when upon storage in sodium phosphate buffer (pH 6.5) at 4C for 5 months. These data collectively confirm that VLPs are reasonably stable and implicate the potentials of EV71 VLPs as a vaccine product.

並列關鍵字

baculovirus virus-like particle EV71

參考文獻


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