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研究生:朱書宏
研究生(外文):Chu, Shuhung
論文名稱:以斑馬魚卵產製新穎抗高血壓胜肽
論文名稱(外文):Production Of The Novel Anti-hypertensive Peptide By Oocytes Of Zebrafish (Danio Rerio)
指導教授:黃尉東
指導教授(外文):Huang, Weitung
口試委員:黃尉東唐品琦張雲祥
口試委員(外文):Huang, WeitungTang, PinchiChang, Yunshiang
口試日期:20120724
學位類別:碩士
校院名稱:大葉大學
系所名稱:分子生物科技學系碩士班
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:85
中文關鍵詞:高血壓β-酪蛋白抗高血壓胜肽吳郭魚卵巢細胞株斑馬魚
外文關鍵詞:hypertensionanti-hypertensive peptidetilapia ovarian cellszebrafishoocytes
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高血壓(hypertension)疾病至今為國人十大死因之一,同時亦是罹患心血管疾病之風險因子。然隨消費者預防保健意識之提升,控制血壓相關機能性食品與類藥物營養劑之需求已日益增加,其中預防高血壓之血管緊縮素轉化酶抑制胜肽(angiotensin converting enzyme inhibiting peptide,ACEIP)更受矚目。目前國立中興大學生命科學系陳全木教授與大葉大學分子生物科技學系陳小玲教授已自 kefir grain 發酵乳原料中分離出一新穎之抗高血壓胜肽(anti-hypertensive peptide No. 1,AP1),經定序解析後發現此關鍵胜肽為 β-酪蛋白(β-casein)中一特定片段之胜肽,其活性表現亦優於現有市售之調節血壓健康食品產品之關鍵主成分VPP 與 IPP。本論文先前以斑馬魚(zebrafish,Danio rerio)為模式動物產生抗菌蛋白(anti-microbial peptide)平台技術之經驗,針對上述之功能性蛋白生產進行試驗。本試驗將抗高血壓胜肽AP1 基因構築於斑馬 VTG 之啟動子下游,續將之選殖入pAAV-IRES-hrGFP 及 pEGFP-N1 之表現載體,並以吳郭魚卵巢細胞株(tilapia ovary,TO-2 cells)分析其表現,並以顯微注射導入斑馬魚受精卵內,觀察並分析其表現位置。結果顯示於細胞轉染第 48 小時後可觀察到其綠螢光之信號,續以 RT-PCR 與西方墨點反應分析,進一步證實具綠螢光蛋白之表現。SDS PAGE 分析亦顯示 AP1 蛋白之可能表現。另將上述之載體以顯微注射之方式轉置至斑馬魚胚胎中之結果顯示,於注射後 24、72、120 及 168 小時以螢光顯微觀察於卵黃囊或肝臟皆具綠螢光之表現,此親代斑馬魚並可產製具螢光表現之第二子代斑馬魚。統計分析結果顯示,96 顆魚卵中,6 顆具螢光表現並持續表現至受精後 2 個月,可達 6.2%之成功率。因此,本試驗已將 AP1 基因成功轉置於斑馬魚基因組中,後續將可藉斑馬魚為生物反應器產製抗高血壓胜肽(AP1)並進行功能性分析。
In Taiwan, hypertension is one of the ten leading causes of death and is the major controllable risk factor associated with cardiovascular disease. Angiotensin converting enzyme (ACE) inhibitory peptides have attracted particular attention and have been studied widely for their applications to prevent hypertension among the bioactive peptides derived from milk proteins. A novel anti-hypertensive peptide (AP1), which derived from milk protein fermented by a nature symbiotic microbial starter-kefir and islated by Dr. Chen at National Chung-Hsing University and Dr. Chen at Da-Yeh University, has a significant effect on reducing systolic and diastolic blood pressure in spontaneously hypertensive rat. Though AP1 can be produced by E coli. and yeast, its post-translational modification and bioactivity is still not clear. The AP1 fragment (202 bp) was constructed with the promoters of vitellogenin, ovarian tumour or female-specific zebrafish zona pellucida genes into pAAV-IRES-VTG/OTU/ZPC-hrGFP and pEGFP-N1 vectors, and these vectors were transfected into tilapia ovarian (TO-2) cells or microinjected into the zebrafish oocytes to establish the transgenic fish line. RT-PCR or western blot analysis revealed that green fluorescent protein (GFP, 55 kDa) and AP1 peptide (8 kDa) could be expressed, observed, and detected after transfection or microinjection. Eggs produced by the transgenic fish showed green fluorescence and lasted to 2 months post fertilization. Only 6 eggs of 96 eggs (6.2%) from founder showed green fluorescence, and whose bioactivity would be further studied. The application of the platform techniques can be an alternative for the development of blood pressure controlling health food or even pharmaceuticals, and can be patented for commercial purpose or for the purpose of biosafety and biomedical researches.
封面內頁
簽名頁
中文摘要................................iii
英文摘要................................v
致謝................................vi
目錄................................viii
圖目錄................................xiii
表目錄................................xv
附錄................................xvi
1. 前言................................1
2. 文獻討論................................3
2.1 高血壓(Hypertension)疾病................................3
2.2 血管緊縮素轉化酶(Angiotensin converting enzyme)與血壓調控之相關性................................3
2.2.1 血管緊縮素轉化酶抑制劑(Angiotensin converting enzyme inhibitor)之簡介..........................4
2.2.2 抗高血壓胜肽(Anti-hypertensive peptide)之簡介................................5
2.3 卵巢腫瘤(Ovarian tumor, otu)基因之簡介................................6
2.4 斑馬魚卵黃蛋白前質基因(Vitellogenin, vtg)之簡介................................7
2.5 斑馬魚雌性專一性透明帶基因(Female-specific zebrafish zona pellucida, zpc).........................7
2.6 斑馬魚(Zebrafish, Danio rerio)之簡介................................8
2.7 轉 殖 基 因 斑 馬 魚 作 為 生 物 反 應 器 之 應用................................9
2.8 外源蛋白之攝取及應用................................9
2.9 研究目的................................10
3. 材料方法................................12
3.1 試驗材料................................12
3.1.1 斑馬魚................................12
3.1.1.1 斑馬魚之飼養................................12
3.1.1.2 斑馬魚之產卵(Breeding)................................12
3.1.2 抗高血壓胜肽(Anti-hypertensive peptide No. 1, AP1)................................13
3.1.3 載體(Vector)................................13
3.2 試驗方法................................13
3.2.1 質體製備................................13
3.2.2 嵌入體片段(Insert fragment)之製備................................14
3.2.3 限 制 酶 酵 素 切 割 反 應 ( Restriction enzyme digestion................................14
3.2.4 連接反應(Ligation)................................14
3.2.5 小量質體之純化................................15
3.2.6 大量質體之純化................................15
3.2.7 勝 任 細 胞 之 製 備 ( Preparation of competent cells)................................17
3.2.7.1 勝任細胞之轉形................................17
3.2.8 瓊脂膠體(agarose)製備與電泳分析................................17
3.2.8.1 1.5 瓊脂膠體製備................................18
3.2.8.2 瓊脂膠體電泳分析................................18
3.2.9 膠體萃取(Gel extraction)................................18
3.2.10 聚合酶連鎖反應(Polymerase chainreaction, PCR)................................ 19
3.2.11 構築載體之定序................................20
3.2.12 顯微注射(Microinjection)................................20
3.2.12.1 顯微注射針之製備(Preparation of injection needles)................................20
3.2.12.2 顯微注射(Microinjection)................................20
3.2.13 注射卵之螢光顯微鏡觀察................................21
3.2.14 細胞培養................................22
3.2.14.1 細胞株之培養條件................................22
3.2.14.2 細胞株之繼代培養................................22
3.2.14.3 細胞轉染................................23
3.2.15 西方墨點(Western blot)分析................................24
3.2.15.1 細胞蛋白質之萃取................................24
3.2.15.2 蛋白質定量................................25
3.2.15.3 配製 SDS-PAGE 膠體................................25
3.2.15.4 SDS-PAGE 分析................................25
3.2.15.5 電轉印(Electroblotting)................................26
3.2.15.6 西方墨點反應(Western blot)................................26
3.2.16 細胞 RNA 之萃取................................27
3.2.17 反轉錄聚合酶連鎖反應(Reverse transcription polymerase chain reaction,RT-PCR)........28
3.3 統計分析................................29
第四章 結果................................30
4.1 質體 pAAV-VTG-AP1 及 pEGFP-N1-AP1 之構築................................30
4.1.1 抗高血壓胜肽(AP1)構築於 pAAV 及pEGFP-N1 之表現載體................................30
4.1.2 質體 pAAV-VTG-AP1 及pEGFP-N1-AP1 之驗證................................ 30
4.3 抗高血壓胜肽(AP1)及 VTG 啟動子基因於pAAV 載體之序列分析................................31
4.4 抗高血壓胜肽基因質體之功能活性分析................................31
4.4.1 TO-2 細胞轉染................................31
4.4.2 反轉錄聚合酶連鎖反應(RT-PCR)及西方墨點反應(Western blot)之分析................................31
4.4.3 Coomassie Brillant Blue 染色法分析................................32
4.5 斑馬魚卵顯微注射之分析與觀察................................32
4.6 斑馬魚第一子代基因轉殖分析................................33
第五章 討論................................34
第六章 結論................................37
參考文獻................................66
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