於桿狀病毒表現系統中建立以2A相似序列發展之多基因表現載體

Develop the evaluation system for 2A-like sequence identification in Baculovirus expression system and apply for establishment of polycistronic baculovirus expression vectors

陳裕傑(Yu-Jie Chen)

指導教授 ： 吳宗遠

中原大學/工學院/生物科技研究所/碩士(2009年)

https://doi.org/10.6840/cycu200901013

摘要

在後基因體時代，不論是蛋白表現系統，基因療法或是疫苗的生產，多基因表現載體 (polycistronic vectors) 的研發是一極重要的課題。桿狀病毒表現系統具有與哺乳類細胞相似的轉譯後修飾作用且產量大、成本低的優勢，目前已廣泛應用於生產重組蛋白。本論文測試源自榕樹透翅毒蛾小RNA病毒 (Perina nuda virus；PnV) 基因體內的兩個2A相似序列；PnV 2AI與PnV 2AII在桿狀病毒表現系統的活性，再利用此2A相似序列發展桿狀病毒多效基因表現載體，並和其他多效基因表現策略做比較，分析在桿狀病毒表現系統內，可做為多效基因表現的最佳策略。首先我們利用分泌性鹼性磷酸脢-綠螢光蛋白融合蛋白(secretory alkaline phosphatase-EGFP fusion protein, SEFP) 在Sf21細胞株中綠螢光分布與無分泌性訊號胜肽(secretion signal peptide) 之EGFP不同，且可藉由螢光顯微鏡分辨的特性，建構出一可非常簡易判斷2A相似序列活性的觀測方法。結果顯示榕樹透翅毒蛾小RNA病毒基因體內的兩個2A相似序列皆可於桿狀病毒感染的Sf21細胞株中表現自我加工胜肽活性，其中PnV2AI較PnV2AII的活性為佳。在雙效基因表現載體中，PnV2AI使胜肽键分開的能力 (ribosomal skipping cleavage efficacy) 在桿狀病毒所感染的Sf21細胞株中約有80%左右，而和其他表現策略相比，其所連接 (flanked) 的上下游基因皆有最好的表現量及活性；另外，在三效基因表現載體的實驗中，結果中顯示，以PnV2AI和PnV539 IRES的組合為構築表現元件所的最佳組合策略，據此我們發展出一個可以在桿狀病毒表現系統中同時表達三基因的表現載體pBac-mcs1-2A-mcs2-Pnir-EGFP。我們已成功利用此載體分別生產含有comma-α次單元及分別含有FSHβ、LHβ及兩個次單元的龍膽石斑魚促性腺激素FSH及LH。

關鍵字

2A相似序列 ； 多效基因表現載體 ； 核醣體內轉譯子 ； 榕樹透翅毒蛾小RNA病毒 ； 桿狀病毒表現系統

並列摘要

In the postgenomic era, the development of polycistronic expression vectors become an important issue for gene therapy, vaccines and heterologous oligomeric proteins production. The baculovirus-insect expression system is recognized as an excellent tool for producing recombinant proteins, hundreds of foreign genes have been expressed by it under the control of a polyhedron promoter. In this study, we have tested the activity of the 2A-like sequences, PnV2AI and PnV2AII, derived from Perina nuda virus (PnV) in baculovirus expression system. First, we took the advantage of the green fluorescence of secretory alkaline phosphatase-EGFP fusion protein (SEFP) in infected Sf21 cells were different with EGFP that didn’t contain secretion signal and could be easy to recognize the 2A-like sequence cleavage efficacy under the fluorescence microscopy. We showed that the two 2A-like sequences from PnV could work in recombinant baculovirus infected Sf21 cells, and the cleavage efficacy of PnV2AI is better than PnV2AII. The bicistronic vector that contained PnV2AI in baculovirus infected Sf21 cells, and protein activity and expression level of the first and secondary genes are better than other bicistronic expression approaches, including the fusion, IRES or two promoters used in our analysis. Second, we constructed tricistronic expression vectors combined the 2A-like sequencs with IRES. In our results, the tricistronic expression vector containing PnV2AI and PnV539 IRES is the optimal combination to express three genes simultaneously. So we developed a tricistronic expression vector pBac-mcs1-2A-mcs2-Pnir-EGFP, to express three genes in a vector in baculovirus expression system. And expressed the gonadotropin of Epinephelus lanceolatus LH and FSH that included the same subunit common-α and two different subunit of LHβ and FSHβ in baculovirus infected Sf21 cells.

並列關鍵字

Perina nuda virus ； IRES ； 2A-like sequence ； polycistronic expression vector ； baculovirus

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