芋頭α-半乳糖水解酶基因的轉殖與表現

淡江大學機構典藏 > 理學院 > 生命科學研究所 > 學位論文 > Item 987654321/51801

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题名:	芋頭α-半乳糖水解酶基因的轉殖與表現
其它题名:	Gene cloning and expression of taro α-galactosidase
作者:	李皇毅;Lee, Houng-yi
贡献者:	淡江大學生命科學研究所碩士班簡素芳
关键词:	芋頭;α-半乳糖水解酶;基因轉殖;基因表現;Taro;α-galactosidase;gene cloning;gene expression
日期:	2010
上传时间:	2010-09-23 16:09:35 (UTC+8)
摘要:	α-半乳糖水解酶（α-galactosidase）是一種可以將末端半乳糖水解的酵素，在各種生物體中都存在，並且擁有許多不同的生物功能，例如在植物中α-半乳糖水解酶參與醣類的代謝和運輸（source to sink transport）。在應用方面，α-半乳糖水解酶可以使B型紅血球表面抗原上的半乳糖水解而變成O型紅血球，因此藉由基因工程的方法取得基因並利用 Pichia pastoris 的蛋白質表現系統來產生大量的芋頭 α-半乳糖水解酶以進行血行轉換的測試和應用。由每公斤的芋的塊莖中進行萃取可獲得約 83.3 Unit 的 α-半乳糖水解酶酵素活性，接著進行三次的管柱層析步驟使蛋白質純化 6019 倍，並使樣品最終純化至單一蛋白質片段，最後樣品的比活性為 54.18 Unit/mg。接著將片段送去進行蛋白質 N 端序列的鑑定和蛋白質中間部分片段序列的鑑定，並利用鑑定的序列和 GH27 family α-半乳糖水解酶的高度保留序列設計 degenerate primer dFP IA 和 dRP 2A 來夾出第一段的基因序列；利用已知的基因序列設計 forward 和 reverse gene specific primer 並經過數次的 PCR 之後夾出完整的基因序列，芋頭 α-半乳糖水解酶的基因由 1082 個 base 所組成，在經過轉譯後其蛋白質分子量為 40.01 kDa。最後將芋頭 α-半乳糖水解酶的基因接入 pPIC9k 質體中，並轉殖至 Pichia pastoris（GS115、SMD1168）中進行基因的誘導與表現。在 Pichia pastoris 中誘導基因表現六天後，GS115 和 SMD1168 的細胞內酵素活性最多分別為 0.5 和 0.45 Unit/mL，而分泌至細胞外的酵素活性則只有胞內的 1/10。 α-Galactosidase is capable of hydrolyzing terminal non-reducing galactosyl residues, and it is distributed in most organisms with many different biological funtions such as metabolism and transportation (source to sink transport) of photoassimilates in plants. In application, α-galactosidase can be used to hydrolyze the galactosyl group of type B antigen on the red blood cell surface, and covert the type B red blood cell into type O. Thus, the Pichia patoris expression system was used to produce large amount of taro α-galactosidase fastly to proceed the seroconversion of erythrocyte. The crude taro α-galactosidase was extracted with 83.3 Unit/kg of enzyme activity from taro tubers. The crude extracts then purified to homogeneity by using column chromatography, and the specific activity was 54.18 Unit/mg after 6019 fold purification. Base on the results of N-terminal sequencing, inner peptide determination, and multiple alignment with GH27 family α-galactosidase, degenerated primers dFP IA and dRP 2A were designed to synthesis the first known sequence of taro α-galactosidase gene. Then forward and reverse gene specific primers were designed from the known sequence to obtain the whole gene sequence, which was consisted of 1082 bases, and the molecular mass of this enzyme was 40.01 kDa, by PCR techniques. The gene was cloned into pPIC9k, and then transformed into Pichia pastoris (GS115 and SMD1168). At last, Pichia pastoris was induced and expressed recombinant taro α-galactosidase. After the recombinant gene had been induced for six days, the intracellular enzyme activities of GS115 and SMD1168 were 0.5 and 0.45 Unit/mL(culture) at most, and the extracellular enzyme activities were only 1/10 fold of the intracellular activities.
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