基因轉譯合成聚胜肽及其應用之探討

Abstract

基因轉譯工程是近年來頗受歡迎的研究熱點之一，學者們設計出許多不同基因序列編碼，利用微生物將基因序列轉譯重組生產出特定聚胜肽或是蛋白質，並研究其相關功能性後加以應用。有學者則將聚胜肽(蛋白質)與合成高分子進行結合，創造出具有特殊功能的新穎生物高分子材料。 本研究主要目標在將聚胜肽與合成高分子進行共聚合得到由天然與人工聚合物所組成的嵌段共聚物。本實驗利用基因轉譯合成目標重組蛋白，再經由凝血酶切位作用得到6kDa目標重組聚胜肽，並再與合成高分子進行共聚合，以獲得聚胜肽高分子產物。在預聚物部分選用PEG-250 Diacid以及Jeffamine® D2000寡聚物，利用這兩個寡聚物個別帶有雙酸及雙胺官能基，進行縮合聚合反應脫水以結合形成胜肽鍵。透過80°C以及EDC兩種合成方式將6kDa聚胜肽與PEG-250 Diacid以及Jeffamine® D2000寡聚物共聚合，合成出聚胜肽-PEG-D2000多嵌段共聚物。 本篇研究結果顯示，經由西方墨點法與核磁共振儀(NMR)分析鑑定聚胜肽產物，確實得到6kDa聚胜肽；並利用FTIR確定PEG-250 Diacid與Jeffamine® D2000能夠順利反應生成胜肽鍵；最後經由His-tag螢光抗體染色，確定順利合成具有聚胜肽之多嵌段共聚物。 本研究成功將聚胜肽與高分子透過共聚合方式進行結合，獲得擁有聚胜肽的多嵌段共聚物。對於未來如何將具有功能性之聚胜肽與高分子進行結合起到了一定的幫助。

In recent years, Translation Engineering has become popular. Many researchers have devised different genetic codes in order to use microorganisms to produce specific recombinant polypeptides or proteins for further study on their functions and application. Some researchers combined the polypeptides (proteins) and synthetic polymers together to create novel biopolymers. The objective of this study was to copolymerize polypeptides and polymers together to obtain polypeptide-co-polymers that consisted of natural and artificial polymers. In the experiment, we used target gene translation to produce a recombinant target protein; then we used thrombin cleavage to acquire 6kDa target polypeptides. After that, we copolymerized 6kDa polypeptides with the synthetic polymer to obtain polypeptide-polymers. PEG-250 Diacid and Jeffamine® D2000 oligomers were selected as prepolymers, these two oligomers of individual diacids and diamines were then used to form peptide bonds by condensation polymerization reactions. Through 80°C and EDC two synthesis methods, 6kDa polypeptides combined with PEG-250 Diacid and Jeffamine® D2000 oligomers were polymerized into peptide-PEG-D2000 multiblock copolymers. The results showed that 6kDa polypeptides were identified by Western blot and nuclear magnetic resonance (NMR); the peptide bond, which was formed by both PEG-250 Diacid and Jeffamine® D2000, was determined by FTIR. Finally we stained a specific label (His-tag) of 6kDa polypeptides with the His-tag fluorescent antibody and determined that the synthesis of polypeptides multiblock copolymers was successfully. This study successful confirms the combination of polypeptides and polymer binding by copolymerization can obtain multiblock polypeptide-co-polymers. For the future, this study will play some useful methods to combine functional polypeptides and polymers.