林慶文臺灣大學：畜產學研究所陳彥伯Chen, Yen-PoYen-PoChen2007-11-292018-06-292007-11-292018-06-292004http://ntur.lib.ntu.edu.tw//handle/246246/63621血管緊縮素轉化酶 ( angiotensin converting enzyme；ACE ) 為人 體調控血壓之重要酵素，在臨床治療上，抑制ACE的活性可治療高血壓。本試驗之目的有二：其一，試驗酒精發酵乳克弗爾抑制ACE之體外試驗；其二，將源於牛β-酪蛋白之ACE抑制肽以基因工程表現於大腸桿菌。 試驗一：將克弗爾粒分別接種於牛乳及羊乳中，試驗其於發酵期間、4℃貯藏期間、熟成條件及固形物添加對ACE抑制能力之影響。試驗結果顯示，牛及羊乳克弗爾於發酵期間之ACE抑制能力皆不佳，但是在貯藏期間兩者之ACE抑制能力皆有提升。發酵完畢後保留克弗爾菌元做不同時間的熟成，再貯藏於4℃，發現兩者之ACE抑制能力較未熟成者強效，且羊乳克弗爾於發酵3天後再貯藏3天時具最強效之ACE抑制能力。 試驗二：將源於牛β-酪蛋白之ACE抑制肽FFVAP以合成基因之方式表現於大腸桿菌。所合成之基因以轉譯為兩個FFVAP為目標設計，且以自行接合之方式將合成之基因單體串成多倍體。將多倍體基因構築於質體pQE31中後轉形至大腸桿菌DH5ㄐA並以聚合酶連鎖反應做轉形株之篩選及倍數之判斷。結果顯示，本試驗成朮c築可轉譯兩及三個ACE抑制肽FFVAP之重組質體，並轉形至表現宿主大腸桿菌M15[pREP4]中。Angiotensin converting enzyme (ACE) is a key enzyme on blood pressure regulation. Inhibition of ACE can be used for antihypertensive therapy, and many clinical antihypertensive drugs have been designed as ACE inhibitor. One purpose of this study is to investigate the ACE inhibitory activity of kefir during different storage and ripening periods. Another object is to produce precursor type recombinant ACE inhibitory peptide in Escherichia coli. Experiment I was conducted to assay ACE inhibitory activity of alcoholic fermented milk, kefir, in different media and conditions. Kefir was fermented by inoculating kefir grains (starter) in bovine and capric milk. We investigated the optimal storage and ripening time for fermenting kefir exhibiting the maximum ACE inhibitory activity. The results showed that ACE inhibitory activity was weak in both bovine and capric kefir during fermentation. However, when kefir were stored at 4℃ for 1 to 7 days, ACE inhibitory activity was improved. When ripened with kefir grains at 4℃ and then stored for various periods, the ACE inhibitory activity was more strong. The capric kefir with 3-day ripening and 3-day storage reached the maximum ACE inhibitory activity. These results suggested kefir exhibited antihypertensive activity in vitro. Further study will focus on the purification of the ACE inhibitory substances and the antihypertensive activity of kefir in animal model. Experiment II produced prodrug type recombinant ACE inhibitory peptide in E. coli. We used synthesized complementary oligonucleotide annealed to make artificial ACE inhibitory peptide gene monomer which can translate to two copies of ACE inhibitory peptide of Phe-Phe-Val-Ala-Pro. In order to produce prodrug type recombinant protein carried many copies of ACE inhibitory peptide, we ligated each monomer of ACE inhibitory peptide gene. The multimer gene was ligated with pQE31 vector and transformed to E. coli strain DH5壹、中文摘要………………………………………………………….1 貳、緒言…………………………………………………………………2 參、文獻檢討……………………………………………………………3 一、血管緊縮素轉化酶抑制肽與抗高血壓肽…………………3 (一)血管緊縮素轉化酶於高血壓之重要性…………………3 (二)血管緊縮素轉化酶與血管緊縮素轉化酶抑制劑…...……..5 (三)食品中之血管緊縮素轉化酶抑制肽與抗高血壓肽……...10 (四)乳品中之血管緊縮素轉化酶抑制肽與抗高血壓肽…...…22 (五)提高血管緊縮素轉化酶抑制肽與抗高血壓肽利用性之方法..…………………………………………………………..28 (六)基因工程與抗高血壓肽……………………………………31 二、大腸桿菌之異源蛋白質表現系統……………………………41 (一)大腸桿菌之簡介…………………………...........................41 (二)大腸桿菌用於異源蛋白質之大量表現……………...……41 (三)大腸桿菌異源蛋白質表現系統之缺點……………….......42 (四)影響異源蛋白質於大腸桿菌表現之因素………...............43 肆、試驗一 酒精發酵乳克弗爾抑制血管緊縮素轉化酶之能力……49 一、前言……………………………………………………………49 二、材料與方法……………………………………………………50 (一)克弗爾之製備………………………………...……………50 (二)乳清之分離.………………………………………………..51 (三)貯藏試驗………………………………………………….. 51 (四)固形物之添加……………………………………………...51 (五)熟成試驗…………………………………………………...52 (六)血管緊縮素轉化酶活性抑制試驗……………………….52 三、結果與討論…………………………………………………..54 (一)克弗爾於發酵期間抑制血管緊縮素轉化酶之能力……..54 (二)克弗爾於貯藏期間抑制血管緊縮素轉化酶之能力……..56 (三)添加固形物之克弗爾於貯藏期間抑制血管緊縮素轉化酶之能力………………………………..……………………..61 (四)不同時期熟成之克弗爾於貯藏期間抑制血管緊縮素轉化酶之能力…………………………………………..………..65 伍、試驗二 重組血管緊縮素轉化酶抑制肽於大腸桿菌之表現……70 一、前言…………………………………………………………..70 二、材料與方法……………………………………………………72 (一)含重覆血管緊縮素轉化酶抑制肽之肽片段抑制血管緊縮素轉化酶之能力……………………………………………72 (二)血管緊縮素轉化酶抑制肽FFVAP2基因多元體之製備….76 (三)構築血管緊縮素轉化酶抑制肽FFVAP2基因多元體於質 體pQE31…………………………………………………..82 (四)含血管緊縮素轉化酶抑制肽FFVAP2基因多元體之大腸 桿菌篩選…..………………………………………………86 (五)含血管緊縮素轉化酶抑制肽基因之重組質體轉形至大腸桿菌表現菌株……………………….…………………….90 三、結果與討論……………………………………………………92 (一)合成肽FFVAPFFVAPFFVAPFFVAP抑制血管緊縮素轉 化酶之能力…………………………………………………92 (二)不同蛋白酶作用於FFVAPFFVAPFFVAPFFVAP對其血 管緊縮素轉化酶抑制能力之影響…………………………94 (三)血管緊縮素轉化酶抑制肽FFVAP2基因多元體之製備….96 (四)構築血管緊縮素轉化酶抑制肽FFVAP2基因多元體於質 體pQE31……………………………………………………98 (五)含血管緊縮素轉化酶抑制肽FFVAP2基因多元體之大腸 桿菌篩選…………………………….…………………….101 陸、結論……………………………………………………………….108 柒、參考文獻………………………………………………………….109 捌、英文摘要…………………………………………………………..126en-US克弗爾重組血管緊縮素轉化酶抑制肽血管緊縮素轉化酶kefirangiotensin converting enzyme inhibitory peptideangiotensin converting enzymethesis