腸病毒七十一型(EV-A71)的感染，特別是在年幼小孩，會引起手口足症並可能造成致命的神經疾病與心肺併發症。此病毒含正股單股RNA，可轉譯一條含有4個結構(VP4-VP2-VP3-VP1)及七個非結構蛋白的前驅多胜肽蛋白。藉由病毒蛋白酶2A及3C的切割，最初產成的VP4-VP2-VP3-VP1前驅結構蛋白可被再切割成VP0 (VP4+VP2), VP3, 及VP1，並在病毒組裝時才漸熟成為4個蛋白組成。一個組裝後的病毒，VP1-VP3暴露於表層，而VP4埋於內部與病毒RNA接合以穩定內鞘結構。VP4在各腸病毒基因型是保守性的蛋白，並在病毒進入細胞後的解鞘過程具有穿膜特性，形成囊泡之膜孔以護行病毒RNA的運輸。理論上，對VP4具有專一結合力的短片段核酸適體 (Aptamer)可用來當作病毒感染的追蹤探針或抑制性阻劑，此篇研究我們即利用系統性配體演化指數擴增法(SELEX)以發展如此之DNA適體以符未來偵測及治療腸病毒之所需。首先細菌質體可編碼產出氮端或炭端有六倍組胺酸標籤(6xHis tag) 的VP4或VP0 重組蛋白被組構以做表達、溶解性，及鎳珠純化的測試。在轉型大腸菌Rosetta II後, 發現pET30b系列質體有較佳的炭端重組蛋白(VP4-His或VP0-His)產成及後續可溶性產物在特定純化步驟後的回收。然而在小規模培養及純化測試中，大約僅有60%重組蛋白可溶於細菌裂解液且少於20%可被鎳珠結合後被競爭液置換到溶液中。因VP4-His比VP0-His有稍高的回收率, 多批次產成被混合以再次通過快速蛋白質液相層析(FPLC)進行純化。然而僅有少量VP4-His 被FPLC高度純化回收，因為混和濃縮的前處理即伴隨VP4-His沉澱。甚且高純度VP4-His 也易於低溫保存時沉澱。因此初始鎳珠結合的VP4-His (VP4珠)，在過度洗除非專一性之細菌蛋白後，被權宜性的運用於SELEX來篩選專一性適體。 接著，含有隨機核甘酸的DNA適體庫被合成，在經過飽和吸附含有外源 6xHis的細菌裂解的鎳珠的負篩選步驟後, 殘留的DNA適體庫被進一步用來進行SELEX的正篩選步驟. 經由選殖每偶數輪SELEX其VP4珠上吸附的DNA適體並定序每次選殖時隨機50個選殖株，有六個適體具有逐漸增加的發生率。我們利用了ELONA的技術比較了六個適體對病毒VP4的結合效果。此研究提供了數個對腸病毒VP4有專一結合力的適體當作潛力性的偵測試劑 。未來在干擾性治療上，則需要更深度了解細部之候選適體最小序列之藥物動力學及其有效度，來限制病毒感染並避免產生有害的免疫反應。

VP4 is a structural component of Enterovirus A71 capsid protein that chaperon viral RNA genome during infection. As short nucleic acid aptamers that specifically target VP4 may act as tracking probes or blocking inhibitors to EV-A7l infection, we aim to develop such aptamers through a Systematic Evolution of Ligands by Exponential Enrichment (SELEX) process. A pET30b-derived plasmid expressing C-terminal 6xHis-tagged VP4 (VP4-His) had a better yield and recovery of soluble VP4-His than the other constructs. However, no more than 60% of VP4-His were soluble in a bacterial lysis buffer and less than 20% can be recovered from VP4-His bound Ni-beads (VP4-beads) in a small-scale purification scheme. Because VP4-His tended to precipitate during concentrating and prolonging low-temperature preservation, further FPLC purification of pooled VP4-His eluates had only poor recovery. Thus, VP4-beads after extensive wash were strategically used in SELEX to select aptamer candidates from a DNA library of approximately 1013 random pool of degenerated oligoes. After negative selection of the library with Ni-beads that were saturated with 6xHis-containing bacteria lysates, the recovered aptamers were enriched for 10 rounds of iterative SELEX selection with VP4-beads. Through cloning of aptamer eluates from VP4-beads collected at even rounds of SELEX and subsequent sequencing of 50 colonies per cloning, we report here that 6 aptamers were identified with increasing high occurrence rates. Affinity comparison of VP4-His to bind these aptamer candidates by an ELONA technique revealed that aptamer-3 has the best potential to be developed as a diagnostic or therapeutic reagent.

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