建構 RNAi 表現質體用於抑制登革熱病毒 NS2 及 NS4

Abstract

登革熱是由蚊子傳播的疾病，主要分布在熱帶與亞熱帶地區。全球大約有 25 億人口生活在病毒傳播疫區，超過 100 個國家有爆發過登革熱。因此，對於登革熱的治療與預防成為目前的重要議題。登革熱屬於黃質病毒科。其病毒基因是由全長 10.7kb 的單股正形 RNA 組成。病毒 RNA 利用單一開放式讀架（open reading fragment）轉譯出三個結構蛋白（C, prM and E）及七個非結構蛋白（NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5）。除了NS2B/NS3 （protease）與 NS5 （RNA-dependent RNA polymerases; RdRp）的酵素功能較明確，其他病毒蛋白在病毒複製與致病機制中扮演的角色則不是非常明確。最近一些研究，推測 NS2A、NS2B、NS4A 及 NS4B 可能與參與病毒複製及造成感染後細胞膜重組有關。為了解這四個病毒基因在病毒複製過程中的功能，本研究嘗試用 RNA 干擾（RNA interference, RNAi）的方式降低調控病毒複製。在本研究中利用六個 siRNA 設計平台軟體針對病毒基因預測具有潛力的 siRNAs（small interfering RNAs），取其交集的結果，建構表現 siRNA 的質體並在 BHK-21 細胞中（baby hamster kidney cell）測試對於病毒複製的影響。在空斑試驗中，短暫性表現 siRNA 的細胞與穩定表現 siRNA 的細胞株皆沒有看到顯著降低複製的結果，表示利用綜合多個設計平台軟體預測出的 siRNA 無法有效地沉默病毒的複製。

Dengue viral infection is a mosquito-borne viral disease in the tropical and subtropical regions. Globally, 2.5 billion people live in over 100 endemic countries and areas where dengue viruses can be transmitted; therefore, the treatments and prevention to the disease become a imperative issue. Dengue virus is a member of the Flaviviridae family. The viral genome consists of a single-stranded, positive-sense RNA of 10.7 kb. The viral RNA encodes three structural proteins (C, prM and E) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5) in a single open-reading-frame. Except for enzymic activities contained within NS2B/NS3 (protease) and NS5 (RNA- dependent RNA polymerases; RdRp), the exact roles of the those proteins in virus replication and pathogenesis are not well defined. In recent studies, NS2A, NS2B, NS4A and NS4B are implicated in assisting viral RNA replication and inducing membrane alteration. In order to understand the functions of those four viral genes in the viral replication, I attempted to down regulate the replication of Dengue virus by the use of RNA interference (RNAi). In this study, I targeted those four viral genes with small interfering RNAs (siRNAs) selected by six siRNA design tools. The plasmids that expressed those siRNAs were constructed and tested in BHK-21 cel (baby hamster kidney cell) to determine the effect on dengue viral replication. According to the results of the plaque assays, there was no significant reduction of viral replication in the cells either transiently or stably expressing the siRNAs. This suggested that the siRNAs designed through combination of those siRNA design tools did not effectively silence the viral replication.