評估昆蟲PnV病毒基因體上的IRES與2A-like sequence在哺乳動物細胞做雙效表現載體的可行性

Development of Bicistronic Expression Vector using Internal Ribosome Entry Sites (IRESes) and 2A-like sequences from the Genome of Perina nuda virus (PnV) in Mammalian cells

陳以希(Yi-Hsi Chen)

指導教授 ： 吳宗遠

中原大學/工學院/生物科技研究所/碩士(2010年)

https://doi.org/10.6840/cycu201001043

摘要

中文摘要
2A是一段多胜肽序列，其作用是當轉譯進行時會有核醣體跳耀(ribosome skipping) 的現象，而生成出兩個蛋白。這段序列可用來在單一個轉譯架構 (opening reading frame, ORF) 連接多個蛋白的基因序列並表現出多個蛋白。最早發現於小RNA病毒 (piconavirus) 的2A胜肽序列與在其他哺乳動物或昆蟲病毒發現的2A相似序列 (2A-like sequence)，已廣泛地應用在生物科技與生物醫學研究上。先前研究證實源自於榕樹透翅毒蛾小RNA病毒 (perina nuda picorna-like virus, PnV) 基因體中的兩個2A相似序列 (PnV2AI與PnV2AII) 可用於昆蟲桿狀病毒表現系統 (baculovirus expression vector system)中並發展多效桿狀病毒表現載體。然而，2A相似序列是否能應用於哺乳動物系統中仍不清楚。在本研究中，我們藉由雙效表現載體可同時表現兩種蛋白質之方法來分析於不同哺乳細胞中，源自於榕樹透翅毒蛾2A相似序列的轉譯活性並比較其他不同雙效策略，如核醣體內轉譯子 (internal ribosome entry site, IRES) 與DNA融合等之活性差異。當在哺乳動物細胞中轉染藉由同義融合(in-frame fusion)方式連接紅螢光 (DsRed) 或分泌性鹼性磷酸酶 (SEAP) 與綠螢光(EGFP)之表現載體時，我們觀察到螢光在細胞內的分佈皆呈現甜甜圈狀(donut-shape)，然而若是以PnV 2A相似序列與IRESes之方式來調控表現下游綠螢光蛋白，其螢光則分佈於整顆細胞。根據以上的觀察，我們認為源自於昆蟲病毒PnV 2A相似序列自我切割 (self-cleavage) 之序列於哺乳細胞中是具有活性的。同時，我們也利用了SEFP報導基因 (SEAP與EGFP融合蛋白之基因) 於不同哺乳細胞中來定量PnV2AI與PnV2AII的活性。其結果顯示，於不同哺乳細胞中PnV2AI不僅比PnV2AII具有更高的活性並比IRESes有更穩定的活性表現。未來，我們期望可以利用本研究所建立的雙效表現載體用於快速篩選和定量更多來自不同病毒的2A-like sequence。

關鍵字

核醣體內轉譯子 ； 核醣體跳耀 ； 2A相似序列 ； 榕樹透翅毒蛾小RNA病毒 ； 雙效表現載體

並列摘要

Abstract
2A is an oligopeptide sequence mediating a ribosome “skipping” effect, producing two non-overlapping proteins. This sequence can be facilitated to connect sequences encoding several proteins into a single open reading frame. 2A (identified in picornaviruses) and 2A-like sequences (found in other mammalian and insect viruses) have become widely used in biotechnology and biomedicine. In previous studies, perina nuda picorna-like virus (PnV) derived 2A-like sequences including 2AI and 2AII were successfully used in the baculovirus expression vector system. However, whether these sequences can be applied in mammalian system is unclear. In this study, we examined the translation activity of 2A-like sequences derived from PnV in different mammalian cells and compared their activity with other approaches, like internal ribosome entry site (IRES) and DNA fusion that used in bi-cistronic vectors to express two genes simultaneously. We found that a donut-shaped fluorescence distribution pattern in mammalian cells transient transfected with plasmids carrying either the red fluorescent protein (DsRed) or secretory alkaline phosphatase (SEAP) and enhanced green fluorescent protein (EGFP) reporter genes joined by an in-frame fusion linker. In contrast, the fluorescence distribution pattern was homogenous in cells transfected with expression vectors in which the downstream EGFP gene is produced independently by way of PnV 2A-like sequences as well as IRESes. Based on these observations, we suggest the self-cleavage 2A-like sequences from PnV, an insect virus, can also function in mammalian cells. Furthermore, we also employed the SEFP reporter gene (a EGFP and SEAP fusion gene) to quantify the activity the PnV 2AI and PnV 2AII in different mammalian cells. Our results indicated PnV 2AI was not only more effective than PnV 2AII but also more stable than IRESes. In the future, we suppose that virus-derived 2A-like sequence in a bi-cistronic expression vector might be rapidly screened and quantified.

並列關鍵字

Internal Ribosome Entry Sites (IRESes) ； ribosome skipping ； perina nuda picorna-like virus(PnV) ； Bicistronic Expression Vector ； 2A-like sequence

參考文獻

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