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  • 學位論文

以多步驟之管柱層析法純化克沙奇A16型類病毒顆粒純化之製程

Purification of Coxsackievirus A16 virus-like particles using multistep chromatography

指導教授 : 胡育誠

摘要


克沙奇A16型病毒為引發常見的手口足病,大多數感染5歲以下孩童並會造成神經上的傷害甚至有致死的可能性。近年來,克沙奇A16型病毒與腸病毒71型經常在西太平洋地區爆發,並造成嚴重的公共健康問題。儘管,由克沙奇A16型病毒所引發的手口足病,病狀較為輕微,但仍然有數例嚴重的病例發生。所以在克沙奇A16型病毒盛行的地區開發有效的疫苗是非常迫切的。此外,由於腸病毒71型的多變性,多價疫苗亦是未來急需發展的。 克沙奇A16型類病毒顆粒與克沙奇A16型真實病毒具有相似的蛋白外殼,且能有效地引發人體的免疫反應。過去本實驗室已成功建構能生產克沙奇A16型類病毒之桿狀病毒。利用P1蛋白切割CD3蛋白成VP0、VP1和VP3,並重組類病毒顆粒。過去實驗室中以建立能純化出高純度之類病毒顆粒之製程,然而,卻無法提高其回收率以及放大純化製程。 因此,本研究中,我們利用改良過後的切向流過濾製程作為純化製程中的第一步,即可去除掉超過88%的雜蛋白,並能回收到超過80%的目標蛋白(克沙奇A16型類病毒顆粒),後續我們利用陰離子交換樹脂TOYOPEARL GigaCap DEAE-650M進行下一步除化,最後我們再使用,疏水性管柱作為最後一步純化,經過濃縮後,我們可以得到純化高達82%,且回收率約為12.8%之純化製程,在純度可以達到理想的結果,在回收率方面更遠高於過去本實驗室中建立的純化製程。未來,可以利用本研究中所得到的高純度、高回收率之克沙奇A16型類病毒顆粒應用於動物之臨床實驗。

並列摘要


Coxsackievirus A16 (CV-A16) is one of the major pathogens associated with hand, foot and mouth disease (HFMD) in infants and young children. In recent years, CV-A16 and human enterovirus 71 (EV-71) have often circulated alternatively or together in the Western Pacific region, which has become an important public health problem in this region. HFMD caused by CV-A16 infection is generally thought to be mild and self-limiting. However, recently several severe and fatal cases involving CV-A16 have been reported. Due to the lack of specific medication and vaccines against CV-A16, there is currently a need to develop effective antivirals to better control CV-A16 infections in epidemic areas. In addition, since prevalent EV-71 subgenotypes change, developing bivalent vaccines composed of the epidemic EV-71 subgenotype and CV-A16 is desirable CV-A16 virus-like particles (VLPs) are empty capsids consisting of viral structural proteins and can elicit potent immune responses, thus holding promise as an CV-A16 vaccine candidate. However, an efficient, scalable production and chromatography-based purification process is missing. To tackle this problem, in this study we first constructed a new recombinant baculovirus co-expressing CV-A16 structural polyprotein P1 and protease 3CD, which can self-assemble into VLP and released into the cultured supernatant. We next modified the tangential flow filtration (TFF) process to include concentration and diafiltration of culture supernatant containing the VLPs which enabled the removal of >88% of proteins while recovering >80% VLPs. We also tested several ion exchange chromatography column resins in different combinations. Using the optimal condition, the concentrated VLPs could be purified using TOYOPEARL GigaCap DEAE-650M resin operated in flow-through mode. After buffer exchange, the VLPs were further purified by a hydrophobic interaction chromatography resin (HIC) as a polishing step and concentrated/diafiltered by an ultra-centrifugal concentrator. The integrated process yielded an overall VLPs recovery of 12.8% and a purity of 82% which were comparatively better than the recovery and purity for the purification of live CV-A16 virus particles. This process thus may move the CV-A16 VLPs vaccine one step closer to the clinical applications.

並列關鍵字

CV-A16 VLP vaccine purification baculovirus

參考文獻


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