- 學位論文
結合切向流過濾與Con A親和性管柱層析法純化昆蟲桿狀病毒載體
Combination of Tangential Flow Filtration and Concanavalin A Chromatography for the Purification of Baculoviral Vector
摘要
桿狀病毒已在近年來開始應用於基因治療的載體，因此我們希望在基因治療上發展一套便利的程序純化桿狀病毒。我們選擇最適合病毒存在的緩衝液條件，再利用切向流過濾（tangential flow filtration, TFF）將病毒環境溶液置換為緩衝液，最後結合TFF和Con A（Concanavalin A）親和性層析法來純化出高純度的桿狀病毒。TFF是個很省時的超過濾系統，且可以移除部份雜蛋白，回收率約在75%，系統對病毒活性也不會造成損失；而Con A親和性層析法是利用methyl-alpha-D-mannopyranoside沖提病毒，回收率大約有21%，純度方面達到90%以上，純化後病毒也可以維持完整性。這種新建立的桿狀病毒純化方法除了能有高純度，所得到的回收率也遠比先前所用的超高速離心（<1%）和固定化金屬親和性層析（1~2%）來的高，除此之外，使用Con A親和性層析也是一種迅速方便而且容易大量純化的方法，這是第一個利用結合切向流過濾和Con A親和性層析來純化桿狀病毒的方法。
並列摘要
Baculovirus is capable of transducing mammalian cells with high efficiency and has been proved to be a promising vector for in vivo or ex vivo gene therapy. In the context of vaccine and gene therapy applications, high purity of baculovirus is necessary, thus we aimed at developing a simple and rapid purification process. In this study, we used a recombinant baculovirus with Vesicular Stomatitis Virus Glycoprotein（VSV-G）on the viral surface, which has been shown to enhance viral transduction efficiency. We also demonstrated that the pseudotyped viral vector can improve viral stability. In order to develop a simple and rapid purification process for recombinant baculovirus, we used tangential flow filtration（TFF）and Concanavalin A（Con A）affinity Chromatography in series for high purity purification. The former is a convenient method to remove host cell proteins and concentrate recombinant baculovirus prior to chromatographic step with a higher recovery of 75%, and the latter leads to approximately 21% recovery of recombinant baculovirus by elution with methyl-alpha-D-mannopyranoside. The newly developed method resulted in an overall recovery yield（~15%）that was higher than those resulting from immobilized metal affinity chromatography（1~2%）and gradient ultracentrifugation（<1%）. In addition, rapid and large-scale purification and high recovery yield of baculovirus can be achieved using Con A affinity chromatography. This is the first report describing the method of TFF combined with Con A affinity chromatography for baculovirus purification.