腸病毒71型(EV 71)隸屬於微小病毒科，是目前已知的六十幾種的腸病毒中致病力最高且最易引起神經性併發症的一種，對於五歲以下的幼童更具有高致死率。然而，目前卻仍然沒有一個有效預防腸病毒71型的疫苗藥物問世。先前本實驗室已經證實以桿狀病毒/昆蟲細胞表現系統所生產出的類病毒顆粒能夠引發動物體內良好的抗體反應，且對多種不同株的腸病毒71型皆具有保護力，顯示類病毒顆粒有潛力作為腸病毒71型的疫苗候選者。因此，在本研究中，我們將致力於大量生產及純化類病毒顆粒。首先，我們著眼於類病毒顆粒大量生產的探討。實驗發現將昆蟲細胞株更換成High Five可提高產量，且類病毒顆粒多分佈胞外。藉由篩選三種常用的無血清培養基後，我們發現使用ESF921培養基能使類病毒顆粒的總產量有效提高，可達20.5 mg/L。另一方面，我們嘗試建立大量純化類病毒顆粒的流程。我們使用PEG沈澱法並結合膠體過濾層析法進行純化。PEG沈澱的操作簡易且省時，對於去除雜蛋白質也有不錯的效果，回收率約為57%。而經膠體過濾層析法純化後的類病毒顆粒，最後的總回收率約10%，純度則約16.5%。這種新建立的類病毒顆粒純化方式所得到的回收率遠比先前的超高速離心(<1%)來得高，是一種省時且容易大量純化的方式。此外，我們更進一步瞭解經PEG沈澱及膠體過濾層析法純化後的類病毒顆粒在免疫效果上面的表現。與經超高速離心純化所得的類病毒顆粒相較之下，由新建立的生產及純化流程所得到的類病毒顆粒也一樣具有良好的誘發體內抗體反應的能力，顯示作為腸病毒71型疫苗確實有其潛力。

Enterovirus 71 (EV71) causes significant morbidity and mortality in child under 5 years of age, but there is no effective vaccine or diagnostic reagent for EV71 to date. Our group has previously demonstrated that virus-like particles (VLP) produced from the baculovirus/insect cell (Sf-9) 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 improve the production and purification processes of EV71 VLP. We found that High Five cells resulted in a significantly higher VLP yield than Sf-9 cells and importantly, the VLP was secreted into the medium. By comparing the VLP expression level in different media, it was found that the yield in High Five cells cultured in ESF921 medium is the highest and can amount to 20.5 mg/L. The VLPs secreted into the medium were precipitated by polyethylene glycol (PEG) with a recovery of □57%. The VLPs were further subject to size exclusion chromatography purification, which resulted in an overall purity of 16.5% and a recovery of 10%. The purified VLPs were subsequently used for immunization of mice and elicited potent antibody responses. These data altogether demonstrate the potential of VLPs produced in High Five cells and purified by size exclusion chromatography as an EV71 vaccine candidate.

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