臺灣博碩士論文加值系統

萊克多巴胺(Ractopamine) 歸類於 β2-腎上腺素受體激動劑(β2-adrenergic agonist)，發現它能夠增加動物肌肉，因此被使用為飼料添加劑，但藥物殘留檢驗也成為食品安全之重要議題。若運用針對特定分子具有高度親和的單株或多株抗體於樣品中所含萊克多巴胺之檢驗，需要投入許多時間及昂貴經費。

本研究 購入以噬菌體呈現技術將外源胜肽表現於M13噬菌體的 pIII 外套蛋白上，以此建構多樣性的隨機胜肽庫，再經由生物親和性篩選(bio-panning)的方法，從中篩選出可與目標物具有專一性結合的胜肽序列；接著利用 ELISA 做為檢測方法，希望藉由此技術可以降低成本，達到快速檢測的目標。首先合成目標物 Ractopamine- bovine serum albumin (RAC-BSA)，再與噬菌體呈現胜肽庫(phage display peptide library)進行三次生物親和性篩選，此當中藉由不斷提高 TBST(Tris-buffered saline Tween-20) 之濃度增加噬菌體對於目標物之專一性與結合強度；接下來去除可能與 BSA 親和之噬菌體後，隨機挑選出 47 個編號之噬菌體進行擴大與純化後，再進行 ELISA 測試，在與 BSA 對照組的結果發現，其中有 19 個對於目標物之吸光值有高於 20 % 之差異，而定序結果顯示有兩組類似序列，第一組為 phage 13(TVRTSAD)、phage 36(TVLREAT)與 phage 49(TVKSYAD)，第二組為 phage 39(LKLGEKW) 與 phage 45(LALQGDS)。最後將此兩組噬菌體與 Ractopamine antibody 進行競爭型 ELISA，結果證明所篩選出來的噬菌體競爭力最好的是 phage 36，加入 phage 36 與抗體競爭之吸光值，比只有 Ractopamine antibody 的對照組下降約 88 %。

Ractopamine is classified into β2-adrenergic agonist. It is found that the animal muscle can be increased, and therefore are used as feed additives, but the drug residue detection has become an important issue of food safety. It is necessary to devote a lot of time and high cost to the use of monoclonal antibody or polyclonal antibodies against a particular molecule for the detection of ractopamine contained in the sample.

In this research, the foreign peptide was expressed on the pIII coat protein of M13 phage by phage display technology to construct a diverse random peptide library that was purchased from New English Biolabs Inc. Then bio-panning was performed to select the peptide sequence that can specifically bound to the targeted object; Furthermore, ELISA was used as a detection method to achieve rapid detection of the target in order to reduce the costs with this technology. First of all, the targeted object ractopamine-BSA was synthesized, and three cycles of bio-panning were conducted with phage display peptide library, by keep on raising the concentration of TBST (Tris-buffered saline Tween-20) to increase the specificity and binding strength of phage displayed peptide to the targeted object. Secondly, the phage displayed peptides that may bind to BSA were removed, and then ELISA was conducted after the random selected forty seven phages were amplified and purified. In the results, compared with BSA control group there are nineteen numbers of phage showed that absorbance of the targeted objects have higher than 20% differences. The results of peptide sequences presented two similar sequences groups, first group are phage 13 (TVRTSAD), phage 36 (TVLREAT) and phage 49 (TVKSYAD). The second group are phage 39 (LKLGEKW) and phage 45 (LALQGDS). Finally, the two groups of phages and ractopamine antibody were conducted by competitive ELISA, the results indicated that phage 36 has the best competitive ability among these selected phages. The absorbance value of the addition of phage 36 to compete with antibody, which was reduced about 88% than that of the control group with only ractopamine antibody.

目錄 I
表目錄 IV
圖目錄 V
中文摘要 1
英文摘要 3
第一章 前言 5
1.1 瘦肉精 5
1.2 萊克多巴胺 (Ractopamine) 6
1.3 萊克多巴胺之使用規範 7
1.3.1 各國與國際組織之使用規範 7
1.3.2 萊克多巴胺於台灣之使用規範 8
1.4 萊克多巴胺之檢測方法 10
1.5 噬菌體呈現技術 11
1.5.1 噬菌體 (Bacteriophage) 11
1.5.2 M13絲狀噬菌體 12
1.5.3 噬菌體呈現技術 (Phage Display Technology) 13
1.5.4 噬菌體呈現系統與抗原決定位 14
1.5.5 噬菌體呈現技術於檢測上之應用 15
1.6 研究目的 16
第二章 材料方法 17
2.1 實驗材料 17
2.1.1 藥品試劑 17
2.1.2 儀器裝置 18
2.1.3 噬菌體呈現胜肽庫(Phage Display Peptide Libraries) 18
2.2 實驗方法 19
2.2.1 生物接合 (Bio-conjugation)－合成偶聯物Ractopamine-BSA (RAC-BSA) 19
2.2.2 噬菌體篩選技術 24
2.2.3 以噬菌體檢測 RAC-BSA 27
2.2.4 競爭型ELISA 28
第三章 結果 30
3.1 RAC-BSA conjugate 分析 30
3.1.1 RAC-BSA 接合 30
3.1.2 RAC-BSA 蛋白質定量 30
3.1.3 SDS PAGE 31
3.1.4 西方墨點法 (Western blot analysis) 31
3.1.5 RAC-BSA 之接合比－Beer’s Law 31
3.2 噬菌體篩選技術 33
3.2.1 RAC-BSA與噬菌體呈現胜肽庫之生物親和性篩選 33
3.2.2 去除會與 BSA 親和之噬菌體 33
3.3 酵素連結免疫吸附分析法 (ELISA) 34
3.3.1 單一溶菌斑挑選與 RAC-BSA 之 ELISA 結果 34
3.3.2 噬菌體 DNA 抽取 34
3.3.3 噬菌體呈現外源胜肽之一致性序列分析 35
3.4 競爭型ELISA 36
3.4.1 Ractopamine antibody 與親和性篩選 phage 競爭目標物 RAC-BSA 之結果 36
第四章 討論 37
表 42
圖 50
附錄 61
參考文獻 73


表目錄
表1 RAC-BSA偶聯物 42
表2 UV全波長掃描鑑定表 43
表3 BSA 標準曲線與 RAC-BSA 蛋白質定量 44
表4 RAC-BSA與噬菌體呈現胜肽庫之生物親和性篩選 45
表5 去除與 BSA 親和之噬菌體 46
表6 核酸測定結果 47
表7 生物親和性篩選後之噬菌體呈現胜肽序列 49

 
圖目錄
圖 1實驗整體流程圖 50
圖 2 RActopamine – BSA 接合之流程 51
圖 3 Ractopamine – BSA 接合之結構圖 52
圖 4 BSA 標準曲線 53
圖 5 RAC-BSA conjugate 之蛋白質電泳 (SDS PAGE) 54
圖 6西方墨點法分析 RAC-BSA conjugate 55
圖 7生物親和性篩選(Bio-panning) 56
圖 8單一溶菌斑挑選與Ractopamine-BSA 之ELISA結果 57
圖 9 ELISA 吸光值相對差異分析圖 58
圖 10噬菌體 DNA 之電泳圖 59
圖 11 競爭型 ELISA 60

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