以桿狀病毒表現系統表現立百病毒(Nipah Virus) 表膜F、G之蛋白研究

Expression of Nipah virus F and G protein using Baculovirus insect system

張詔棋(Erick Putra Theophilus)

指導教授 ： 吳宗遠

中原大學/理學院/生物科技研究所/碩士(2019年)

https://doi.org/10.6840/cycu201900861

摘要

立百病毒（NiV）為副黏液病毒屬，其宿主是狐蝠屬的果蝠，是一種高致病性的人畜共通病毒，會引發呼吸問題與腦神經病變，如在人類中引起急性熱病腦膜炎。此病毒的首次爆發在1998年的馬來西亞，多年來以印度病例最多，目前市場上還沒有可用的NiV疫苗。NiV F和G蛋白位在病毒表面，在病毒進入宿主細胞時扮演重要角色，本研究嘗試以生產NiV的融合蛋白（F）和糖蛋白（G），以篩選出能防止NiV和宿主膜的融合的藥物或抗體，未來可應用於防止病毒的感染和爆發。我們使用簡單和安全的桿狀病毒bac-to-bac蛋白表現系統來表達NiV F和G蛋白。首先，我們設計F蛋白的C端帶有his tag作為蛋白的標的，並以IRES共表現螢光蛋白作為病毒篩選的標的，已成功重組完成此雙效表現桿狀病毒，在西方墨點法中，可看見66 kDa的F0 與45 kDa的F1蛋白片段。之後，再將G蛋白基因接入含有F蛋白基因的三效表現載體，重組共表現F、G與螢光蛋白的桿狀病毒，即可最為抗原生產與藥物或抗體的篩選平台。

關鍵字

立百病毒（NiV） ； 立百病毒融合蛋白（F） ； 立百病毒糖蛋白（G） ； 雙效表現質體 ； 桿狀病毒 ； 桿狀病毒表現系統 ； bac-to-bac系統

並列摘要

Nipah virus (NiV) is a virus belonging to paramyxovirus which reservoir host is fruit bats of genus Pteropus. This virus is a highly pathogenic zoonotic paramyxovirus that can cause a respiratory problem and known to attack the brain of the animals such as causing acute febrile encephalitis in human (Chua et al, 2000). The first outbreak of this virus happens in Malaysia 1998 which India has the most cases over the years. Currently, there still no available vaccine of NiV in the market which this research try to achieve by producing the Nipah Fusion (F) protein and Glycoprotein (G). The NiV F and G protein are essential to the virus as the tools for the virus to enter the host cells by fusing the virus cell with the host cells walls while it is activated. The NiV F and G proteins are located on the surface of the virus which makes these proteins important as the drugs or antibodies target to prevent the fusion of both NiV and the host membrane which will prevent the infection and preventing any outbreak from this virus in the future. The easiest and safest way to express the NiV F and G protein is by using bac-to-bac system which utilizes the Baculovirus as the vector to carry both of NiV F and G genes to the insect cells to be expressed. The first step to express the recombinant gene is to create the plasmid which contains the F protein. After the plasmid that contains NiV F successfully synthesize, the F gene then will be express using bac-to-bac Baculovirus-insect cells system. After that, the F will be delivered to the new plasmid that contains two region sites which can simultaneously express both NiV F and G proteins with also using bac-to-bac Baculovirus-insect cells system. The result of protein expression can be tested using Western Blot techniques by comparing the size of the proteins and sub proteins produced by the Sf21 cells.

並列關鍵字

Nipah Virus (NiV) ； Nipah Virus Fusion Protein (F) ； Nipah Virus Glycoprotein (G) ； Bi-cistronic Plasmid ； Baculovirus ； Baculovirus-Insect Expression Vector ； Bac-to-bac System

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