- 學位論文
生產重組人面蜘蛛絲蛋白之研究
Production of recombinant spider silk proteins from Nephila Pilipes
摘要
蜘蛛是已存在於地球上數百萬年的生物，且數量龐大種類繁多，蜘蛛可藉由蜘蛛絲進行捕食、求生和繁衍。蜘蛛絲是由蛋白質所構成的纖維，是已知的天然高性能材料，蜘蛛可產生出許多種絲，且具有不同特性，如良好的強度、彈性、抗菌性及生物相容性，因此於複合材料、生物醫學等方面均有應用潛力。由於蜘蛛飼養及取絲不易，無法有效的大規模生產，可行的方法是應用重組DNA技術，將基因轉殖入其他的蛋白質表達系統，並透過生物資訊技術分析胺基酸序列和遺傳密碼，尋找及設計可以達成目標功能性蛋白質分子，期望得到與蜘蛛絲相似的生物纖維。蜘蛛絲的蛋白質組成包含了三個部分：Ｎ端（N-terminal）、Ｃ端（Ｃ-terminal）的保守序列區（conserved region）以及中間的重複序列區（repetitive region），Ｎ端及Ｃ端的序列影響蛋白質成絲，重複序列區使蜘蛛絲具有強度及彈性，此實驗以人面蜘蛛(Nephila pilipes) 的大壺狀腺(major ampullate silk)所分泌的曳絲 (MaSp1) 為目標，取得蜘蛛絲片段基因，再利用限制酶酵素組裝，結合兩端的保守序列區以及片段重複序列區，組合成與天然蜘蛛絲相似的生物材料，並嘗試利用大腸桿菌(Escherichia coli)及嗜甲醇酵母(Pichia pastoris)進行生產，並建立一個有效生產的平台。以使用帶有表現稀有密碼子(rare codon)載體之大腸桿菌Rosetta 2 (DE3)進行搖瓶生產，結果顯示僅有小片段重組蜘蛛絲蛋白質成功表達，而嗜甲醇酵母Pichia pastoris KM71H表達系統則有待後續進一步調整。
並列摘要
Spiders have existed on earth for million years. There are a large number of spiders, and a wide variety of spider species are found in the world. Spiders use silk to hunt, wrap up their prey, suspend themselves away from dangers and protect their offspring away from predators. The spider silk is a kind of protein fiber which is spun by spiders, also it is a kind of well-known high performance material. Spiders can produce many specific types of silk characteristics suitable for different broad properties, for example, high strength, elasticity, antimicrobial resistance and biodegradable ability. Therefore, it has potentiality to be used as a complex and biocompatible material. Because of spiders are not easy to carry out livestock breeding and it is hard to collect silks from living spiders, spider silks could not be an efficient and large-scale production. In order to overcome these problems, it could be taken advantage of recombinant DNA technology to expresses natural spider silk genes into different host organisms. To get spider silk-like biomaterial, it is available to utilize the bio-information analyzing amino acid sequence and gene coding, which could search and design the target of functional protein molecular. Spider silk protein is consisted of three different domains： the conserved region of the N-terminal domain, the conserved region of C-terminal domain, and repetitive region of the middle portion. N-terminal domain keeps spidroins being soluble in the spider’s glands during storage, C-terminal domain ensures spidroins form well-ordered fibers, and repetitive region makes the fibers strong, elastic and stable. In this study, the goal was to target the spider silk protein (MaSp1) which is secreted from major ampullate silk gland of Nephila pilipes. To obtain and clone MaSpI gene, we conducted PCR to amplify partly MaSpI fragment and restriction enzyme digestions and ligation of the specific MaSpI into plasmids, including the conserved regions and the repetitive region. The recombinant spider silk proteins were expressed by Escherichia coli and yeast Pichia pastoris which are considered as the efficient production platforms. The Escherichia coli Rosetta 2 (DE3) strain was chosen as a candidate of the expression system which has a rare codon pRARE2 plasmid for optimal eukaryotic protein expression. On the other hand, the Pichia pastoris strain KM71H which carries Mut-(methanol utilization) knockout was also selected for spider silk production. Last, the expression results showed that in the E. coli expression system only small molecular weight recombinant spider silk protein fragments were expressed successfully; in the Pichia pastoris KM71H expression system, silk protein expression and secretion efficiency were low, requiring further tune-up and adjustment.