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標題: 利用人造油體系統製備蜂王漿抗菌胜肽royalisin之抗體與王漿抗菌胜肽之二級結構功能探討
Preparation of a polyclonal antibody against royalisin, an antimicrobial peptide from royal jelly of Apis mellifera via an artificial oil body system and characterization of its functional secondary structure
作者: 曾俊銘
Tseng, Chun-Ming
關鍵字: honeybee;下咽頭腺;Apis mellifera;hypopharyngeal gland;royalisin;artificial oil body antigen display system;王漿抗菌胜肽人造油體抗原展現系統
出版社: 生物科技學研究所
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王漿抗菌胜肽(Royalisin)為蜜蜂工蜂下咽頭腺分泌的抗菌胜肽,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為小片段胜肽,為了提高動物對此小胜肽所產生的免疫反應,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肽的能力而產生抗體。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肽及天然蜂王漿中的王漿抗菌胜肽,王漿抗菌胜肽的專一性抗體將有助於王漿抗菌胜肽的研究及特性分析。本研究亦利用人造油體系統純化重組王漿抗菌胜肽,經由DTT處理,探討二級結構對於王漿抗菌胜肽抗菌活性的影響,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.
其他識別: U0005-0308201023404700
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