在桿狀病毒表現系統中建立細胞融合技術以發展抗立百病毒之藥物篩選平台

立百病毒（Nipah virus, NiV）為一高致病性人畜共患病毒，最早於1999年在馬來西亞分離出來。而至今立百病毒已經在世界上感染了650多人，且死亡率高達70%。由於其高致死率與高傳染性，迫切需要開發針對立百病毒感染的治療策略。再者，若需要使用活病毒進行治療對策開發，也較易造成研發人員的感染風險。因此，我們開發了在桿狀病毒表現系統中，利用昆蟲細胞表達立百病毒的膜蛋白，因而造成細胞融合之現象，建立對抗立百病毒之藥物篩選平台。由立百病毒所誘發的細胞融合機制，主要有幾個蛋白質參與，包含NiV融合蛋白 (NiV F)、NiV糖蛋白(NiV G) 、ephrinB2 和組織蛋白酶 L。而其中桿狀病毒與昆蟲細胞皆有組織蛋白酶Ｌ的同源蛋白，因此實驗設計上，我們構築兩種桿狀病毒，一株為同時表達NiV F和NiV G，另一株則單純表現ephrinB2 。利用西方墨點法與和免疫螢光染色分析，我們能成功地在細胞膜上表現NiV F，NiV G 和 EphrinB2，然而，初步實驗卻發現共感染無法造成細胞融合現象。根據先前的研究顯示，在昆蟲細胞中，降低培養基的 pH 值和添加膽固醇可以促進細胞融合的形成。而當我們進一步額外置換較低 pH 值之培養基與添加膽固醇時，的確能成功觀察到 NiV 所誘導的細胞融合。我們利用此平台進一步篩選幾種化合物，其中包括一些多磺化萘胺化合物與蛋白酶抑製劑等。在這些化合物中，蘇拉明（多磺化萘胺化合物）對 NiV表現出最高的融合抑製劑活性。此研究證實我們所建立由 NiV誘導產生的細胞融合平台，可以作為一個安全、簡單的藥物篩選平台，用於尋找具有抗 NiV 融合抑製劑特性的化合物。

關鍵字

立百病毒；桿狀病毒表達載體系統；細胞融合；融合抑製劑；蘇拉明

並列摘要

Nipah virus (NiV) is known as a highly pathogenic zoonotic virus, which was isolated in Malaysia in 1999. To date, NiV has affected more than 650 individuals with high mortality rate, up to 70%. Due to its high pathogenicity and outbreak potency, therapeutic countermeasure against NiV is urgently needed. Therefore, we developed a drug screening platform using baculovirus expression system expressing NiV recombinant proteins. This platform utilized the fusion formation to evaluate the efficacy of a tested compound as fusion inhibitor against NiV. NiVinduced syncytium is orchestrated by several proteins, including NiV fusion protein (NiV F), NiV glycoproteins (NiV G), ephrinB2, and cathepsin L protease. As the baculovirus and insect cells expressed a homologue form of cathepsin L, only two baculoviruses were engineered to express NiV F and NiV G simultaneously, as well as ephrinB2. Despite of the successful expression of those three proteins on insect cell membrane, confirmed by western blot and immunofluorescence assay, the co-infection between those two baculoviruses demonstrated negative result for syncytium formation. However, NiV-induced syncytium was successfully observed in infected insect cells when lower pH and cholesterol was supplemented in the culture medium, as previous studies showed that this condition could enhance the syncytium formation. The platform was further used to screen several compounds, including some phytochemicals, polysulfonated napthylamine compound, and protease inhibitor. Among these compounds, suramin, the polysulfonated napthylamine compound, demonstrated the highest fusion inhibitor activity against NiV. This result suggested that our platform could serve as a safe and simple alternative drug screening platform for finding compound with fusion inhibitor properties against NiV.

並列關鍵字

Nipah virus ； baculovirus expression vector system ； syncytium ； fusion inhibitor ； suramin

參考文獻

1. Bossart, K.N., D.L. Fusco, and C.C. Broder, Paramyxovirus entry, in Viral Entry into Host Cells.

Google Scholar

2013, Springer. p. 95-127.

Google Scholar

2. Chua, K., et al., Nipah virus: a recently emergent deadly paramyxovirus. Science, 2000. 288(5470):

Google Scholar

p. 1432-1435.

Google Scholar

3. Paterson, D.L., P.K. Murray, and J.G. McCormack, Zoonotic Disease in Australia Caused by a

Google Scholar

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在桿狀病毒表現系統中建立細胞融合技術以發展抗立百病毒之藥物篩選平台

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