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  • 學位論文

研究不同啟動子以及IRES在錦鯉魚鰭細胞、哺乳動物細胞以及昆蟲細胞內的活性

Study of the different promoters and virus internal ribosome entry site (IRESs) activity in the Koi fin cells (KFC) , mammalian cells and insect cells

指導教授 : 吳宗遠

摘要


蛋白質表現系統為現今重要的生物技術工具。除了魚細胞系統外,許多表現載體已被開發提供各種表現系統所使用。對於一個完善的表現載體首先必須具有合適的啟動子在特定的細胞中進行下游基因的轉錄表現,因此在本實驗中先測試CMV啟動子 (cytomegalovirus major immediate-early promoter)、Krt4啟動子 (Keratin 4 promoter)、Hsp70啟動子 (Heat shock protein 70 promoter)、Beta-actin啟動子 (β-actin promoter)以及Cmlc2啟動子 (cardiac myosine light chain 2 promoter)在魚鰭細胞、哺乳類細胞以及昆蟲細胞內的活性,而當表現載體具備合適的啟動子後,即可將IRES (internal ribosome entry site)應用在此載體上並建構出由單一個啟動子表現多個外源蛋白的雙效表現載體。實驗結果顯示在哺乳類細胞中CMV啟動子活性較其他啟動子活性高,在中國倉鼠卵巢細胞內CMV啟動子活性約為其他啟動子的15倍,但在魚鰭細胞株CMV啟動子活性與其他啟動子並無明顯差異。另外以雙效表現載體來測試encephalomyocarditis virus (EMCV)、Rhopalosiphum padi virus (RhPV)以及 Enterovirus 71 (EV71)三種病毒的IRES在魚鰭細胞與哺乳細胞內的活性,實驗結果顯示EV71 IRES在哺乳類細胞內活性都比RhPV IRES以及EMCV IRES的活性高,而在魚鰭細胞中也可觀測到EV71與RhPV IRES的活性。

並列摘要


Protein expression systems are important tools in modern biotechnology. Many expression vectors provided for the different expression system to produce protein have been developed, except the fish cell systems. The promoter is one of the elements for the expression vectors that regulate gene expression on transcription level and have specific for different type cells. To finding the promoter that is optimal in the different cells, CMV, Krt4, Hsp70, beta-actin and Cmlc2 promoter was test in Koi Fin cells (KFC), mammalian cells and insect cells. In addition, to produce more than two of proteins in the same vector, dicistronic vector was used for this study. A IRES element was constrstruct into the expression vector to produce the second protein that was translated by a mechanism of cap-independent translation from the same transcript. The IRESs: EV71, EMCV and RhPV IRES were tested in the KFC cells and mammalian cells. The results demonstrate the CMV promoter have strong activity in mammalian cells, especially for the CHO cells and its activity is more than others about fifteen times. Nevertheless, all of the promoters were weak in the KFC by the transient transfection assay. The Hsp70 promoter seemed to be better than other promoters except compare with CMV promoter either in KFC or mammalian cells. In addition, the EV71 IRES activity was stronger than RhPV and EMCV IRES activity in mammalian cells although RhPV IRES as well as EV71 can function well in the KFC.

並列關鍵字

IRES Promoter KFC

參考文獻


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.
林以婷. 2008. 發展非裂解性昆蟲細胞雙效表現載體. 私立中原大學碩士學位論文, 桃園.
滕昭怡. 2006. 於桿狀病毒表現系統中以RhPV 5' UTR IRES同時表現不同胞器標定蛋白特性分析之研究. 私立中原大學碩士學位論文, 桃園.
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.
Baird, G.S., Zacharias, D.A., and Tsien, R.Y. (2000). Biochemistry, mutagenesis, and oligomerization of DsRed, a red fluorescent protein from coral. Proc Natl Acad Sci U S A 97, 11984-11989.

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