登革病毒蛋白酶篩藥平台開發暨抗病毒藥物篩選

Development of a cell-based assay for Dengue Virus NS2B/NS3 protease activity and anti-DENV drug screening

陳韋翰(Wei-Han Chen)

指導教授 ： 李景欽

高雄醫學大學/生命科學院/生物科技學系碩士班/碩士(2015年)

摘要

登革病毒是一種藉由蚊子傳播的病原菌，主要橫行於熱帶與亞熱帶。根據文獻記載，登革病毒每年的感染人數已攀升至三億九千萬人。而在2014年，台灣就已累計一萬五千多的病例。不幸的是目前尚未有核可的登革病毒疫苗以及治療方法。所以開發抗病毒藥物是刻不容緩的。登革病毒非結構性蛋白3（nonstructural protein 3 , NS3），是病毒的蛋白酶， 更是發展抗病毒藥物中很好的標靶。在偵測病毒蛋白酶活性的相關研究測驗法常利用純化的病毒蛋白酶結合人工合成的胺基酸胜肽，但此種方法可能無法反映蛋白酶在活細胞中或是活體內的真實狀態。為了達到能夠在細胞中高通量篩選抗病毒藥物，我們建構了許多報導質體，其中如pEG(△MITA)sNluc。此種質體編碼了由綠螢光蛋白（enhanced green fluorescence protein, EGFP）以及分泌型刺蝦冷光酶secreted nono luciferase (sNluc) 的融合蛋白，中間以病毒蛋白酶的專一性切位作為linker相連。因此我們可藉由偵測分泌至細胞外的報導蛋白活性推知病毒蛋白酶的活性。在本實驗中，我們發現比起表現突變的病毒蛋白酶，偵測出來的相對蛋白酶活性可以達到2.5倍。整體而言，這個細胞內的報導系統提供了一個良好的藥物篩選平台。
在本研究中我們也篩選出化合物E-Guggulsterone，一種源自Commiphora mukul 的guggul萃取物擁有抑制登革病毒複製以及蛋白表現的能力。再者，我們發現E-Guggulsterone能在登革病毒感染的Huh 7細胞中能增加干擾素（interferon）的表現。之後，我們也將更深入的探討其抗病毒機轉。

關鍵字

登革病毒 ； 蛋白酶

並列摘要

Dengue (DEN) is a mosquito-borne pathogen threatening people within tropics and subtropics. Accordingly, there are 390 million dengue infections per year, and 96 million manifest apparently. In Taiwan, it is estimated to be 15 thousand infections since 2014. Unfortunately, neither specific treatment nor vaccine is available for DENV. Therefore, it is urgent to discover anti-DENV agents. DENV nonstructural protein 3 (NS3), a viral protease, is a promising target for anti-DENV agents. Recombinant viral protease combined with synthetic peptides is a common protease activity assay in vitro, however, it can’t reflect the situation in live cells or in vivo. To achieve the requirement for high-throughput screening in a cellular condition, we created several reporter plasmids including pEG(△MITA)sNluc and pEG(△MITA)SEAP. pEG(△MITA)sNluc encodes a fusion protein in which Enhanced Green Fluorescent Protein (EGFP) was fused to secreted nono luciferase (sNluc) through the NS3 protease butapeptide recognition sequence which is cleavable for viral protease. Therefore, the activity of NS2B/NS3 protease can be quantitatively indicated by measuring SEAP or Nluc activity. In our study, we found that the activity of SEAP or Nluc is 2.5 folds comparing to mutant NS3 protease control. Overall, this study provides a cell-based reporter system to facilitate high-throughput screening of NS2B/NS3 inhibitors.
We also show that E-Guggulsterone, a guggul extract from Commiphora mukul, exhibits inhibitory activity on DENV RNA replication and protein synthesis in infectious system. Furthermore, we demonstrated that E-Guggulsterone increases interferon (IFN) expression in DENV-infected Huh 7 cells. In the future, we will elucidate the mechanism of E-Guggulsterone on anti-DENV activity.

並列關鍵字

dengue virus ； protease

參考文獻

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