王漿抗菌胜&;#32957;（Royalisin）為蜜蜂工蜂下咽頭腺分泌的抗菌胜&;#32957;，其由51胺基酸所組成，大小為5.5 kDa，其中有6個半胱胺酸（cysteine） 組成三個分子內的雙硫鍵，使其結構及活性相當穩定。由於royalisin為小片段胜&;#32957;，為了提高動物對此小胜&;#32957;所產生的免疫反應，因此，本研究利用人造油體抗原呈現系統來製備royalisin的抗體。我們利用基因工程分別將royalisin架構到油體膜蛋白質oleosin中間疏水區域的N端及C端，形成royalisin-OleC-royalisin之融合蛋白質，並藉由大腸桿菌表現系統大量表現及純化。因為佐劑含有礦物油，而融合蛋白質與油混合均質化後會形成乳糜球，疏水性油體膜蛋白質中央區則會插入到油質中，並將N端及C端的royalisin展現在油體的表面，形成一個人造油體抗原呈現系統，以此系統製備抗體將有助於增加免疫系統辨識到小片段胜&;#32957;的能力而產生抗體。結果發現，製備出的抗體可專一的辨認到重組的王漿抗菌胜&;#32957;及天然蜂王漿中的王漿抗菌胜&;#32957;，王漿抗菌胜&;#32957;的專一性抗體將有助於王漿抗菌胜&;#32957;的研究及特性分析。本研究亦利用人造油體系統純化重組王漿抗菌胜&;#32957;，經由DTT處理，探討二級結構對於王漿抗菌胜&;#32957;抗菌活性的影響，結果發現雙硫鍵的保留與其抗菌活性有相當的關係。

Royalisin is the small peptides secreted by hypopharyngeal gland of Apis mellifera. It contains 51 amino acid residues with 6 cysteine residues forming three intramolecular disulfide linkages, perhaps resulting in a compact globular structure exhibiting high stability at low pH and high temperature. Since royalisin was small peptide, it is not easy to prepare its corresponding antibody. In this study, we want to prepare a specific antibody against to small peptide royalisin via artificial oil body. Royalisin was constructed to the N- and C-terminal of oleosin central hydrophobic domain as the oleosin fusion protein, royalisin-oleosin central domain-royalisin (Roy-OleC-Roy). Roy-OleC-Roy fusion protein was expressed and purified in E. coli AD494 (DE3). The purified fusion protein was mixed with Freund’s adjuvant to form emulsion and used for the production of antibody in chicken. In this system, oleosin is used as the carrier which possesses a lipophilic segment embedded in the hydrophobic oil core and the royalisin which linking to the two arms protruding on the surface of the oil bodies. In results, we have successfully to prepare the antibodies to against small antimicrobial peptide royalisin by this antigen display system. This antibody can be useful for study and characteristic analysis of royalisin. In this study, we also purified an antimicrobial peptide-royalisin via an artificial oil body system, and treat with the reducing agent dithiothreitol(DTT). As results show the disulfide bonds are very important for antimicrobial activity of royalisin.

中文摘要 i
Abstract ii
目次 iii
壹、緒論 1
1.1 蜂王漿 1
1.2 王漿抗菌胜肽（Royalisin） 2
1.3 油體簡介 3
1.4 油體膜蛋白（oleosin）介紹及其相關應用 4
1.5 抗體製備系統 12
1.6 研究動機 15
貳、材料與方法 17
2.1 油體膜蛋白載體之構築 17
2.2 構築表現載體-pJ01-roy-OleC-roy 18
2.2.1 利用聚合酶連鎖反應（PCR）選殖royalisin序列 18
2.2.2 瓊脂凝膠電泳分析與回收 18
2.2.3 DNA接合作用 19
2.2.4 轉型作用與藍白篩選 19
2.2.5 重組質體之回收 20
2.2.6 構築含有融合蛋白質roy-OleC-roy之表現載體 21
2.3 融合蛋白質Roy-OleC-Roy的表達及純化 21
2.3.1 微生物之大量表現 21
2.3.2 融合蛋白質之純化 23
2.4 人造油體穩定性測試 24
2.4.1 磷脂質與芝麻油的製備 24
2.4.2 人造油體之重組 24
2.4.3 油體的穩定性測試 25
2.5 抗體的製備及回收 25
2.5.1 蛋白質濃度測定 25
2.5.2 多株抗體（Polyclonal antibody）之製備 26
2.5.3 多株抗體之純化與回收 26
2.6 西方墨點法(western blot) 27
2.6.1 Tricine-SDS-PAGE 27
2.6.2 西方墨點法測試 28
2.7 重組王漿抗菌胜肽的純化與抗菌分析 29
2.7.1 融合蛋白質oleosin-inteinS-royalisin表達 30
2.7.2 融合蛋白質（royalisin-inteinS-oleosin）之純化 30
2.7.3 重組王漿抗菌胜肽之純化 31
2.7.4 最低抑菌濃度測試（Minimal inhiboitory concentration, MIC） 32
2.7.5 最低殺菌濃度（Minimal Bactericidal Concentration Assay,MBC） 33
2.7.6 重組王漿抗菌胜肽－Royalisin之蛋白質殺菌動力學分析（Time kill determination） 34
2.7.7 胰蛋白質水解酶對重組王漿抗菌胜肽之影響 35
參、實驗結果 36
3.1 royalisin抗體製備純化與分析 36
3.1.1 攜帶子蛋白質載體之構築 36
3.1.2 構築融合蛋白質(roy-OleC-roy)的表現載體 36
3.1.3 大量表現及純化融合蛋白質（roy-OleC-roy） 37
3.1.4 人造油體的穩定性測試 38
3.2 重組王漿抗菌胜肽royalisin二級結構對抗菌活性的影響 41
3.2.1 重組王漿抗菌胜肽的抑菌及殺菌所需最低濃度測試 41
3.2.2 蛋白質殺菌動力學分析（Time kill kinetic assay） 46
3.2.3 經Trypsin處理後重組王漿抗菌胜肽的最低抑菌濃度測試 48
肆、討論 49
4.1 以人造油體系統作為抗原呈現系統來製備小胜肽的抗體 49
4.2 以人造油體純化系統統純化出具有活性的雙硫鍵抗菌胜肽 50
4.3 重組王漿抗菌胜肽具有開發成為新型抗菌藥物的潛力 51
4.4提高人造油體蛋白質表達及純化系統的回收效率 53
4.5 探討重組王漿抗菌胜肽其雙硫鍵與抗菌活性之分析 54
參考文獻 56
圖表 66
表一、本研究所使用的引子 67
表二、本研究所使用測試抗菌活性之菌種 68
表三、重組王漿抗菌胜肽對11種細菌之抗菌作用 69
圖一、攜帶子蛋白質載體pET-29-OleC之構築 70
圖二、表現載體pJ01-roy-OleC-roy構築之流程圖 71
圖三、SDS-PAGE電泳分析融合蛋白質（roy-OleC-roy）在大腸桿菌AD494之表現 72
圖四、溫度對人造油體之穩定性分析 74
圖五、royalisin抗體對天然蜂王漿專一性之分析 75
圖六、SDS-PAGE電泳分析融合蛋白質（roy-inteinS-Ole）在大腸桿菌AD494之表現 76
圖七、西方墨點法分析由人造油體蛋白質純化系統之重組王漿抗菌胜肽 77
圖八、革蘭氏陽性菌存活率試驗 78
圖九、革蘭氏陰性菌存活率試驗 79
附錄 80
附錄一、油體膜蛋白質oleosin之二級結構圖 81
附錄二、人造油體蛋白質表達及純化系統的構築示意圖 82
附錄三、人造油體抗原展現系統的示意圖 83
附錄四、royalisin抗體之架構示意圖 84

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