選殖與表現牛流行熱病毒C端截短之G和GNS醣蛋白

Cloning and Expression of Bovine Ephemeral Fever Virus’ C-terminally truncated G and GNS glycoproteins

羅亦渟(Lo Yi-Ting)

指導教授 ： 馬丁萊恩 ； 鄭力廷

國立屏東科技大學/國際學院/動物用疫苗國際學院專班/碩士(2018年)

https://doi.org/10.6346/NPUST202000505

摘要

牛流行熱病毒（BEFV）分類上屬於桿狀病毒科，是以節肢動物為媒介感染牛及水牛的急性病毒性疾病－牛流行熱（BEF）的病原體。牛流行熱病毒在澳洲、非洲及亞洲是一個日益嚴重的問題，在爆發期間對經濟產生嚴重影響。

目前有四種牛流行熱疫苗，其中兩種為G蛋白次單位疫苗和重組蛋白疫苗，然製造純化的免疫原成本高昂且不易純化為其缺失。因此亟需新的策略以製造重組G蛋白。我們已使用合成生物學技術，將BEFV具有免疫源性之外膜醣蛋白質G（主要的免疫原）和GNS的C端穿膜區序列刪除，並以具有親合性的V5 抗原標記取代之，使其蛋白質能從細胞分泌至培養液中。本研究已將這些蛋白做免疫點漬法、西方墨點法與免疫螢光術等功能分析，初步結果顯示，C端截短之BEFV-G和BEFV-GNS重組蛋白質能表現並分泌至培養液中，爾後將會用快速有效的方法純化它。

以更簡易的方法製造出具免疫原性的醣蛋白，並且提高這些醣蛋白的產量是本研究的目標，期望此基礎研究是往後相關科學研究領域之基石。

關鍵字

牛流行熱病毒 ； V5 抗原標記 ； C端截短之BEFV-G蛋白

並列摘要

Bovine ephemeral fever virus (BEFV), a member of the family Rhabdoviridae, is the causative agent of the arthropod-borne viral disease of cattle and water buffalo, bovine ephemeral fever (BEF). BEFV is an increasing problem in Australia, Africa and Asia causing serious economic impact during outbreaks. The subunit G protein-based vaccines and recombinant vaccines are two of four types of BEF vaccine however, the methods of these vaccines are difficult to purify and expensive to make it. Therefore, there is demand for a new strategy to produce recombinant G (the major immunogen) proteins.

We used synthetic biology to create a form of the virus membrane-anchored G and GNS glycoprotein with the C-terminal trans-membrane domain deleted and replaced by the affinity V5 epitope tag, creating an immunogen that would be secreted from the cell into the tissue-culture cell media. Functional analysis of these proteins were detected with immuno-dot blot, western blot and immunofluorescence assays. We showed this recombinant, C-terminally truncated form of BEFV G and GNS protein were expressed and secreted into the media. The secreted, V5-tagged, G and GNS protein will then purify from the media in a highly effective, single-step method.

The aim of this project is straight forward to create secreted forms of these immunogenic glycoproteins and maximize the expression level of these glycoproteins. From basic research- molecular biology and protein expression will lead to broadly applicable studies.

並列關鍵字

Bovine ephemeral fever virus (BEFV) ； V5 epitope tag ； secreted G protein

參考文獻

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