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研究生:余鍾蘭
研究生(外文):Chung-Lan Yu
論文名稱:降高血壓胜肽基因之選殖與表現
論文名稱(外文):Cloning and Expression of Antihypertensive peptide gene
指導教授:李根永李根永引用關係
指導教授(外文):Ken-Yuon Li
學位類別:碩士
校院名稱:東海大學
系所名稱:食品科學系
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:81
中文關鍵詞:降高血壓胜肽血管收縮素轉換酶血管收縮素轉換酶抑制劑
外文關鍵詞:Angiotensin-converting enzymeAngiotensin-converting enzyme inhibitorLeu-Arg-Pro
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摘 要
Leu-Arg-Pro為一個在降高血壓方面具有高效力的三胜肽,對於血管收縮素轉換酶(Angiotensin-converting enzyme,ACE)來說,為強力的競爭型抑制物。本研究主要目的是利用DNA重組技術建構一表現系統來生產具有抑制ACE的功能之降高血壓短鏈胜肽。實驗依據此胺基酸殘基序列,設計兩段互補的降高血壓胜肽三重複核苷酸序列(ACEI1 & ACEI2),利用基因重組技術將此核苷酸序列和表現載體pEZZ18接合起來,形成能生產降高血壓胜肽的基因重組載體(pEZZ18-ACEI),再將此基因載體轉型進入宿主細胞E.coli JM105中,以找出能合成降高血壓胜肽的轉型株。篩選出來的轉型株經由液體擴大培養後,利用親和性管柱層析膠體的專一性結合、回收具有降高血壓胜肽的融合蛋白,接著將融合蛋白以chymotrypsin水解、釋放出(Leu-Arg-Pro)3後,利用高效能液相層析法分析降高血壓胜肽之正確性。
Abstract
The tripeptide, Leu-Arg-Pro, possesses efficacy to lower blood pressure, since the peptide is a highly competitive inhibitor of ACE (Angiotensin-converting enzyme). The objective of this study is to construct a expressing recombinant vector which confers the transformed cell to produce an ACEI (Angiotensin-converting enzyme inhibitor) peptide. The gene of the ACEI peptide consisted of two single strand complementary oligonucleotides that encode a peptide with amino acid sequence as (Leu-Arg-Pro)3. The double strand oligonucleotides with Sal I sticky end were inserted into the vector pEZZ18, and then the recombinant plasmid (pEZZ18-ACEI) were transformed into E.coli JM105. Some transformed colonies that were capable to produce a fused protein linking up the ACEI peptide were selected. The ACEI peptide linked with ZZ protein was isolated with an affinity chromatography. The fusion protein was treated with chymotrypsin to release the ACEI peptide. The ACEI peptide was verified with an HPLC system.
中文摘要-----------------------------------------------------I
英文摘要-----------------------------------------------------II
壹、前言------------------------------------------------------1
貳、文獻回顧--------------------------------------------------2
一、血管收縮素轉換酶 (Angiotensin-converting enzyme, ACE) 與血壓的關係------------------------------------------------------2
(一) ACE影響血壓之機制------------------------------------2
(二) ACE之性質--------------------------------------------3
(三) ACE活化位置模型--------------------------------------5
(四) ACE抑制活性測量法------------------------------------7
二、血管收縮素轉換酵素抑制劑(ACE inhibitors, ACEI)降血壓作用--8
(一) ACEI降血壓效果之來源與發現---------------------------8
(二) ACEI作用機制----------------------------------------11
(三) ACEI之設計----------------------------------------------12
三、胜肽合成方法比較-----------------------------------------15
(一)化學合成法-------------------------------------------16
(二)酵素性合成法-----------------------------------------17
(三)DNA基因重組法----------------------------------------18
參、材料與方法-----------------------------------------------19
一、宿主細胞(Host cell)的培養與保存--------------------------19
(一)材料---------------------------------------------------19
1.1 菌種-------------------------------------------------19
1.2 培養基-----------------------------------------------19
1.2.1 LB broth培養基(Luria-Bertina broth)--------------19
1.2.2 LB plate平面培養基-------------------------------19
1.2.3 篩選型培養基(LB/Amp/X-gal agar plate)------------19
(二)儀器---------------------------------------------------20
(三)方法---------------------------------------------------20
3.1 宿主細胞的培養---------------------------------------20
3.2 宿主細胞的保存---------------------------------------21
二、表現載體之製備-------------------------------------------21
(一)材料---------------------------------------------------21
1.1表現載體-----------------------------------------------21
1.2電泳試劑-----------------------------------------------23
(二)儀器---------------------------------------------------23
(三)方法---------------------------------------------------24
3.1勝任細胞之製備-----------------------------------------24
3.2轉型作用-----------------------------------------------25
3.3質體DNA之抽取------------------------------------------25
3.4電泳分析方法-------------------------------------------26
3.5限制酶剪切反應-----------------------------------------27
3.6基因片段之回收-----------------------------------------27
三、ACEI胜肽核苷酸序列之製備---------------------------------27
(一)材料----------------------------------------------------28
1.1 ACEI胜肽核苷酸序列之合成------------------------------28
(二)儀器----------------------------------------------------28
(三)方法----------------------------------------------------28
3.1 ACEI胜肽核苷酸序列之黏合------------------------------28
3.2限制酶截切反應-----------------------------------------29
3.3基因片段之回收-----------------------------------------29
3.4電泳分析-----------------------------------------------29
四、ACEI胜肽基因載體之構築-----------------------------------29
(一)材料----------------------------------------------------29
(二)儀器----------------------------------------------------30
(三)方法----------------------------------------------------30
3.1接合作用-----------------------------------------------30
3.2轉型作用-----------------------------------------------31
3.3重組載體的篩驗-----------------------------------------31
3.4送交定序-----------------------------------------------31
五、重組ACEI胜肽之表現與純化---------------------------------32
(一)材料----------------------------------------------------32
1.1定序確認成功之轉型株-----------------------------------32
1.2一之1.2所述的培養基------------------------------------32
(二)儀器----------------------------------------------------32
(三)方法----------------------------------------------------32
3.1蛋白質的表現-------------------------------------------32
3.2蛋白質濃度測定-----------------------------------------33
3.3硫酸十二酯鈉-聚丙烯醯胺膠體電泳分析(SDS-PAGE)----------33
3.3.1試劑-----------------------------------------------33
3.3.2儀器設備-------------------------------------------34
3.3.3方法-----------------------------------------------35
3.4親和性管柱層析法---------------------------------------36
3.5以chymotrypsin切割融合蛋白-----------------------------39
3.6高效能液相層析法分離-----------------------------------39
肆、結果與討論-----------------------------------------------41
一、ACEI胜肽核苷酸序列之製備-------------------------------41
二、ACEI胜肽基因載體之構築---------------------------------43
三、重組ACEI胜肽之表現與純化-------------------------------53
四、重組ACEI胜肽之分離-------------------------------------58
五、總結---------------------------------------------------63
伍、結論---------------------------------------------------64
陸、參考文獻-------------------------------------------------65
柒、附錄-----------------------------------------------------72
一、NCBI基因庫中之質體pEZZ18相關資料。---------------------73
二、#17轉型株重組載體之DNA定序結果-DNA定序圖譜。-----------78
三、#35轉型株重組載體之DNA定序結果-DNA定序圖譜。-----------80
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