發展非裂解性昆蟲細胞雙效表現載體

Development of bi-cistronic nonlytic insect cell expression system using internal ribosome entry sites

林以婷(Yi-Ting Lin)

指導教授 ： 吳宗遠

中原大學/理學院/化學系/碩士(2008年)

https://doi.org/10.6840/cycu200900207

摘要

桿狀病毒/昆蟲細胞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。然而此表現系統是利用病毒感染作為載具，會造成細胞的裂解，不利於生產需後修飾的分泌性蛋白與膜蛋白。故本論文將發展非裂解性昆蟲細胞表現系統以改善桿狀病毒/昆蟲細胞表現系統的缺失。非裂解性昆蟲細胞表現系統包含暫時的轉染與穩定表現的轉型細胞株兩種，為了要便利挑選轉型成功的細胞株，我們將篩選適合的核糖體內轉譯子（internal ribosome entry site；IRES），以IRES共表現綠螢光基因作為細胞株選殖的標的。首先將秋行軍蟲細胞株Sf21以轉染的方式來快速篩選出適合的IRES，結果顯示PnV539 IRES比RhPV IRES 更合適於此系統。因此我們利用PnV539 IRES共表現綠螢光基因作為快速篩選轉型細胞株的標的，成功建構出三種穩定表現的細胞株：DPnE、SPnE與IFNγPnE，分別可穩定的表現欲表達蛋白，DsRed、SEAP與IFN-γ。另一方面針對PnV539 IRES進行宿主範圍的研究，發現在大部份的昆蟲細胞株中，除源自於果蠅的S2細胞外，PnV539 IRES皆比RhPV IRES 具有較佳的轉譯活性，尤其PnV539 IRES在Sf9的細胞株中具有最佳的轉譯效果，另外有趣的是發現在哺乳類CHO細胞中亦為PnV539 IRES較RhPV IRES有較佳的轉譯活性。為了要探索IRES的結構與功能，我們將PnV539 IRES進行序列剔除的分析，發現在前150個核苷酸對於此PnV IRES非必要的序列，顯示PnV IRES只需5' UTR的323個核苷酸序列與下游ORF的66個核苷酸進行協同作用，即可達到最佳的轉譯效果。

關鍵字

PnV IRES ； 非裂解性昆蟲細胞表現系統 ； 雙效載體

並列摘要

The baculovirus-insect cell expression system (BEVS) is recognized as a valuable tool for recombinant proteins production. The advantages of BEVS include (1) post-translational processing in insect cells is similar to mammalian cell (2) high recombinant proteins production yield and (3) the production costs are lower than mammalian cells based expression systems. However, BEVS used recombinant virus as vectors to infect host cell and eventually resulted cell lysis and compromised most of post-translational processing. Thus, BEVS is unfavorable for secreted and membrane proteins production. To overcome this bottleneck, the thesis focused on the modification of nonlytic insect cell expression. In order to select stable transform cell lines convenient, we screened the suitable internal ribosome entry site (IRES) for co-expression EGFP protein as a selection marker. First, the Sf21 cell line was transient transfect with selective IRESs. The results show that PnV539 IRES is stronger than RhPV IRES in Sf21 cells. Then, we employed PnV539 IRES to co-express the genes of interest with EGFP to facilitate the creation of stable cell lines. We succeed in building the three different stable transformed cells: DPnE, SPnE and IFNγ PnE that can continuous expressing DsRed, SEAP and IFNγ, respectively. These results promoted us to study the host range of PnV539 IRES. We can found out PnV539 IRES has better translation activation than RhPV IRES in most insect cell lines, excepted the Drosophila derived cell line, S2. Especially, PnV539 IRES has the best translation result in the Sf9 cells. Interestingly, we got the same results in the mammalian CHO cells. Finally, we employed deletion assay to explore the structure and function of PnV539 IRES, We showed that 1-4 stem loops including the 5' end first 150 nucleotides were not necessary for the function of PnV539 IRES and suggested the synergistic effect between the 323 nucleotides of 5' UTR and 66 nucleotides of downstream ORF is necessary for the optimal activity of PnV539 IRES.

並列關鍵字

Bi-cistronic vector ； PnV IRES ； Nonlytic insect cell expression system

參考文獻

吳奕珍. 2006. 常蚜病毒5’末端序列在哺乳動物細胞和昆蟲細胞中轉錄和轉譯活性之分析 私立中原大學碩士學位論文,桃園.

陳聖奇. 2007. 不同IRES在昆蟲及哺乳動物細胞株進行轉譯之研究. 私立中原大學碩士學位論文, 桃園.

Ailor, E., and M.J. Betenbaugh. 1998. Overexpression of a cytosolic chaperone to improve solubility and secretion of a recombinant IgG protein in insect cells. In Biotechnol Bioeng. Vol. 58. 196-203.

Ailor, E., and M.J. Betenbaugh. 1999. Modifying secretion and post-translational processing in insect cells. In Curr Opin Biotechnol. Vol. 10. 142-5.

Baird, G.S., D.A. Zacharias, and R.Y. Tsien. 2000. Biochemistry, mutagenesis, and oligomerization of DsRed, a red fluorescent protein from coral. Proc Natl Acad Sci U S A. 97:11984-9.