重組桿狀病毒表現系統目前已被廣泛應用在蛋白生產、疫苗開發及基因治療上，其具有可攜帶大片段外源基因的優點，但已有文獻報導重組桿狀病毒隨著不斷繼代過程中會產生大量的干擾缺損顆粒，造成後代病毒的基因不穩定性並導致重組蛋白的產量減少。目前尚未有研究針對桿狀病毒深入地探討外源片段大小對於重組桿狀病毒基因穩定性的影響，以及其所能夾帶的最大外源片段容量(capacity)。因此本研究的主要目的為探討外源基因片段大小與重組桿狀病毒基因穩定性之間的關聯，並進一步推展至實際應用時，載體中各種不同性質的外源片段對於病毒載體基因穩定性是否造成影響。我們以EGFP (enhanced green fluorescent protein)基因表現匣作為偵測外源片段穩定存在的指標，同時添加不同大小的外源填塞片段 (stuffer)，建構出多株重組桿狀病毒以進行病毒轉導效價及基因DNA定量等分析，深入探討外源片段大小以及病毒代數對於重組桿狀病毒基因穩定性的影響。另外，本實驗室已將長效表現系統Sleeping Beauty (SB)結合桿狀病毒載體應用於癌症的治療且效果顯著，也證實此混成表現系統的應用潛力所在，因此我們在本研究中以SB系統作為基礎平台，插入各種有利於桿狀病毒載體表現的單元因子，在提升SB系統基因表現的同時探討各種因子以及病毒代數對於SB混成重組桿狀病毒基因穩定性的影響。我們發現當外源片段長度增加，第二代病毒仍能維持穩定的外源基因表現，然而隨著病毒代數的提升，其外源片段遺失的現象越明顯，顯示不同大小的外源片段確實會對於重組桿狀病毒的基因穩定性產生影響。另外在混成SB長效表現系統中，將SB100剪切酶表現匣與其轉位子IR/DR置於同一株病毒的建構方式於第二代病毒皆能良好地攜帶外源片段，但隨著病毒連續繼代則明顯較容易產生基因不穩定的現象，我們推測除了外源片段長度的影響之外， CMV啟動子於昆蟲細胞中微量表現的SB100剪切酶也可能致使IR/DR間的egfp表現匣更容易從重組病毒中遭到剃除。這些結果提供實驗室在建構大片段外源基因及SB長效表現系統的重組桿狀病毒時一個基因穩定性的參考。

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