林慶文2006-07-262018-06-292006-07-262018-06-292004http://ntur.lib.ntu.edu.tw//handle/246246/16465牛乳鐵蛋白(bovine lactoferrin)為乳中的 一種運鐵蛋白，其具有很多生理功能。牛乳 鐵蛋白素(bovine lactoferricin)是乳鐵蛋白位 於N 端的一段多肽，其大小約為25 個胺基 酸。在哺乳動物體內，乳鐵蛋白被胃蛋白酶 截切後可得到乳鐵蛋白素，而此片段具有比 乳鐵蛋白更強的抗菌活性，係利用乳鐵蛋白 素作為抗菌物質之開發。本研究旨在探討運 用重組嗜甲醇酵母菌於體外產製乳鐵蛋白素 之可行性。首先利用人工合成不同物種之乳 鐵蛋白素以及乳鐵蛋白素抗菌中心片段之多 肽，並直接對大腸桿菌、金黃色葡萄球菌及 念珠球菌進行抗菌試驗。結果顯示，最佳殺 菌效果之多肽為牛乳鐵蛋白素以及其抗菌中 心片段。挑選牛乳鐵蛋白素25 個胺基酸當中 含11 個胺基酸之抗菌核心片段作為本研究 欲進行產製、分析、試驗之目標片段。將可 轉錄出此多肽之DNA 序列以核酸合成法取 得後，再將目標基因放入酵母菌表現型載體 當中。確定帶有乳鐵蛋白素之質體建構無誤 後，以電穿孔方式將此建構好的質體轉形至 Pichia pastoris 細胞，藉以產製生物性抗菌多 肽。在蛋白質分析部分，藉由Tricine-SDS PAGE 以及西方墨點法之分析，可在膠片上 看到預期大小17 kDa 的色帶。因此確認牛乳 鐵蛋白素於嗜甲醇酵母菌系統當中被表現出 並分泌至細胞外。Bovine lactoferrin is a transferrin protein found in milk, which has many biological functions. Lactoferricin is a short fragment from the N-terminus of lactoferrin. This peptide is about 25 amino acids. Lactoferricin is generated upon gastric pepsin cleavage of lactoferrin in mammalian animals. This peptide is much more effective in antibacterial properties than lactoferrin. And we could use the peptide to develop new style probiotic products. Our study is focus on the generation of the recombinant yeast, Pichia pastoris, containing the bovine lactoferricin gene fragment and the feasibility of large-scale production. First, we got synthetic lactoferricin and it’s antibacterial core peptide of different species by peptide synthesis, and then tested the antimicrobial activities against Escherichia coli, Staphylococcus aureus and Candida albicans. Results revealed that the bovine lactoferricin and its antibacterial core peptide show the better ability of antimicrobial than the others. Then 11-amino acids antibacterial core peptide of bovine lactoferricin were selected, which has the same antibacterial activity as the native lactoferricin. Using oligonucleotide synthesis to get oligonucleotides of desired peptides, and cloned the target gene into yeast-expression vectors. After the constructed bLFcin/pPICZα B vectors were confirmed, we transformed the recombinant vectors into Pichia pastoris by electroporation and selected the transformants to express the biological antibacterial peptides. In protein analysis, an expected protein size of recombinant bLFcin was detected by Tricine SDS-PAGE and Western blot. The results revealed that recombinant bLFcin was successful produced by P. pastoris expression system.application/pdf606673 bytesapplication/pdfzh-TW國立臺灣大學動物科學技術學系暨研究所乳鐵蛋白素抗菌多肽嗜甲醇酵母菌酵母菌表現型載體Lactoferricinantibacterial peptidePichia pastorisyeast expression cassettereporthttp://ntur.lib.ntu.edu.tw/bitstream/246246/16465/1/922313B002134.pdf