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研究生:李長政
研究生(外文):Chang Cheng Li
論文名稱:探討修飾PorcineTeschovirus2A胜肽之胺基酸序列於多基因表現系統中胜肽斷裂效率的影響
論文名稱(外文):Genetic modified amino acid of 2A peptides from Porcine Teschovirus improves peptide cleavage activity in multicistronic systems
指導教授:張國友
指導教授(外文):K. Y. Chong
學位類別:碩士
校院名稱:長庚大學
系所名稱:醫學生物技術研究所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
論文頁數:61
中文關鍵詞:2A 胜肽豬鐵士古病毒多基因表現系統
外文關鍵詞:2A peptidesPorcine Teschovirusmulticistronic systems
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2A胜肽為一個約20 個胺基酸的小片段的序列,最早發現於小RNA病毒 (Picornavirus) 中的口蹄疫病毒 (foot-and-mouth disease virus, FMDV),最近研究也顯示在其他病毒中也擁有跟2A胜肽相似的序列且擁有斷裂的作用。這些胜肽的序列都同樣擁有一段保留區域:-GDxExNPG↓P-。2A胜肽的斷裂作用是核糖體在轉譯時經過2A胜肽時,核糖體則略過其中一個胜肽鍵 (peptide bond)。利用這原理,2A胜肽已經被廣泛的應用在多基因表現系統,該系統可以在載體同時表現多個蛋白且蛋白能擁有相同的分子數。然而,在不同來源的2A胜肽序列卻有著不同的斷裂效率,尤其從 Porcine Teschovirus (PTV)簡稱 P2A ,有著很低的斷裂效率。因此,本研究主要目的為利用基因修飾的方法來改變PTV的2A胜肽胺基酸序列,探討修飾後2A胜肽之胺基酸序列於多基因表現系統中胜肽斷裂效率的影響。 (1)比較不同病毒來源的2A胜肽序列的斷裂效率在多基因表現系統,(2)利用基因修飾的方法修飾或增加PTV的2A胜肽的胺基酸序列對胜肽斷裂效率的影響。研究結果顯示:先前的實驗發現目前現有廣泛被使用的P2A之2A胜肽序列,在螢光顯微鏡下觀察和西方墨點法的結果都明顯顯示其斷裂效率很低。在經過修飾後P2A之2A胜肽的胺基酸序列,發現其斷裂效率和基因表現能力皆有些微增加。另外,在P2A之2A胜肽的胺基酸序列前個別添加兩組不同三個胺基酸後,結果發現斷裂的活性有大幅的提昇。甚至其中一組基因表現能力有顯著增加。由本研究結果證明修飾或添加於P2A之2A胜肽的胺基酸序列會影響斷裂的效率及基因表現能力。期望未來能應用於多基因表現系統的載體上供基因治療使用。
2A peptides is about 20 amino acids small peptides sequence which is first found in the Picornavirus foot-and-mouth disease virus (FMDV). Recent study demonstrated that 2A-like peptides sequences are also found in many other viruses and have ‘cleavage’ activity. These peptides sequence have conserved motif: -GDxExNPG↓P-, It have been shown that ribosome translating via 2A peptides sequence induced ‘skipping’ effect, producing seems a cleavage of polyprotein in mammalian cells. Based upon results, 2A peptides have been widely used as a linker sequence in multicistronic vectors for co-expression multiple proteins at the similar level in a single vector. Despite of its success, these 2A or 2A-like peptides from difference resource have been shown different cleavage activity, especially 2A peptides from Porcine Teschovirus (PTV) has low cleavage activity. Therefore, we employed the genetic modified amino acid of 2A peptides from PTV would improve cleavage activity of polyprotein in mammalian cells. To accomplish our goal, we studied: (1) to compare the cleavage activity of 2A peptides sequences from difference resource in multicistronic vector system, (2) to evaluate the cleavage activity of genetic modify amino acid of 2A peptides from PTV. Our data shown: The 2A peptide from PTV has low cleavage activity, but has a higher gene expression level in our system. Furthermore, we also demonstrated that several genetic modify amino acid of 2A peptides of PTV are increased cleavage activity. In conclusion, we have success to modify amino acid of PTV 2A peptides for higher cleavage efficiency in multicistronic systems. This system would provide us an opportunity to improve usefulness of expression vector for gene therapy in near future.
指導教授推薦書……………………………………………….…………….i
口試委員會審定書……………………………………….…………………ii
授權書…………………………………………………….……………...…iii
誌謝……………………………………………………….……………...…iv
中文摘要…………………………………………………………………….v
英文摘要…………………………………………….……………………...vi
縮寫表…………………………………………….…….………………….vii
第一章 前言………………………………………………………………...1
1. 多基因表現載體的策略…………………………………..………..1
2. 2A 胜肽序列………………………………………………………...3
第二章 實驗設計…………………………………………………..……….7
第三章 材料與方法……………………………………………..………….8
第四章 實驗結果……………………………………………….………….18
1. 建立不同病毒來源的2A胜肽序列連結的多基因表現載體………18
2. 不同病毒來源的2A胜肽序列斷裂效率……………………………18
3. P2A胜肽胺基酸序列修飾後的斷裂效率……………………..……19
4. 增加GPG於修飾P2A胺基酸序列前的斷裂效率…………..……..21
5. 綜合比較增加GPG及GSG於修飾P2A胺基酸
序列後其基因表現活性……………………………………………22
第五章 討論……………………………………...........…………..23
參考文獻……………………………………………………………...……26
圖表……………………………………………………………...…………31
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