不同IRES在昆蟲及哺乳動物細胞株進行轉譯之研究

Identified Internal Ribosome Entry Site for Insect and Mammalian cells

陳聖奇(Sheng-chi Chen)

指導教授 ： 吳宗遠

中原大學/理學院/化學研究所/碩士(2007年)

https://doi.org/10.6840/cycu200700828

摘要

桿狀病毒表現系統（Baculovirus expression system）為近年廣為應用之真核表現系統，而以苜蓿夜蛾核多角體病毒（Autograph californica nucleopolyhedrovirus； AcMNPV）為基礎之重組病毒最廣為使用。與傳統常用之原核及真核表現系統相比，此系統之蛋白質轉譯後修飾較接近於哺乳動物細胞，但其成本則較哺乳動物低廉。為能以桿狀病毒表現系統更有效表現生產多樣化的蛋白質，本實驗室將IRES（Internal ribosome entry site）導入此系統中，建構能以單株病毒表現兩種以上外源蛋白之雙效表現載體。而本篇論文主要希望能了解實驗室開發之PnV539 IRES是否能於IPLB-Sf21以外之昆蟲細胞株表現IRES活性，另外找出EV71、EMCV及RhPV此三種IRES何者是最適合用於哺乳動物細胞雙效表現載體之開發。而研究結果也顯示PnV539 IRES於IPLB-Sf21、NTU-LY-3S、IPLB-BmN4和IPLB-LD652Y等多種昆蟲細胞中皆具有IRES活性，但於哺乳動物細胞中之IRES活性尚無法得知。此外於質體轉染U2-OS、COS-1及CHO細胞發現EV71 IRES其IRES活性較其他兩者為佳。由以上結果顯示PnV539在許多昆蟲細胞中皆具有IRES活性，應是未來開發昆蟲細胞雙效表現系統極佳之選擇。而以EV71 IRES建構之桿狀病毒雙效表現載體，應能成為極具潛力之基因治療工具。

關鍵字

IRES ； 桿狀病毒

並列摘要

Baculovirus expression vectors system （BEVS）that based on Autograph californica nucleopolyhedrovirus （AcMNPV） exten¬sively used for protein expression in recent years. Comparing with traditional Prokaryotic （ex. E. coli） and Eukaryotic （ex. Yeast） expression systems, this system has similar post-translation processing with mammalian cells. To extend the applications of BEVS, we have used internal ribosome entry sites （IRES） to construct bi-cistronic baculovirus expression vectors. In this study we explored the activity of PnV539 IRES in insect cells besides IPLB-Sf21. We were also determine the IRES activity of EV71, EMCV and RhPV IRES in mammalian cells （CHO, COS-1, U2-OS ）. The results suggest PnV539 IRES can express EGFP reporter gene in IPLB-Sf21, NTU-LY-3S, IPLB-BmN4 and IPLB-LD652Y. However, the IRES activity of PnV539 IRES in mammalian cells is still unknown. In transfection assay the activity of EV71 IRES in mammalian cells are better than EMCV and RhPV IRES. These results demonstrate the PnV539 IRES might be a good choice for non-lytic bi-cistronic baculovirus expression vectors. In addition the EV71 IRES might be suitable to construct bi-cistronic baculovirus expression vector for gene therapy.

並列關鍵字

IRES ； baculovirus

參考文獻

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