蜂王漿抗菌胜肽Royalisin之二級結構功能探討

Study the Secondary Structure and Function of Royalisin, an Antimicrobial Peptide from Royal Jelly of Apis mellifera

黃勝昌(Sheng-Chang Huang)

指導教授 ： 彭及忠

國立虎尾科技大學/文理學院/生物科技研究所/碩士(2013年)

摘要

蜂王漿抗菌胜肽(Royalisin)為蜜蜂工蜂下咽頭腺分泌的小分子抗菌胜肽，其由51個胺基酸所組成，分子量大小約為5.5 kDa，含有6個cystein組成的三個分子內雙硫鍵，確保結構、活性穩定表現。研究發現，royalisin對於革蘭氏陽性菌、革蘭氏陰性菌和真菌具抗菌活性，經基因序列比對發現與其它抗菌胜肽不同處在於C端的11個胺基酸，為了研究此11個胺基酸是否影響royalisin之活性表現，本研究利用基因工程架構出一段刪除C端末端的11個胺基酸的DNA序列(royalisin-D)，結合油體膜蛋白質oleosin與內含肽inteinS，形成oleosin- inteinS-royalisin-D之融合蛋白質，利用微生物大量表達融合蛋白質、以人造油體純化系統進行重組蛋白質純化。本實驗亦製備蜂王漿抗菌蛋白質royalisin，除此之外，添加DTT處理純化完之重組蛋白質，藉此比較還原過雙硫鍵之重組蛋白質與未處理的部分進行活性測試比對，利用最低抑菌濃度測試、最低殺菌濃度測試等活性測試方法研究兩種蛋白質之活性表現。結果顯示，重組蛋白質royalisin-D對於Staphylcoccus aureus、Staphylcoccus xylosus、Staphylcoccus intermedius B、Paenibacillus larvae、Pseudomonas aeruginosa等菌株仍具有抗菌效果，但其抗菌能力與royalisin相比無明顯差異，可見蜂王漿抗菌蛋白質royalisin C端末端的11個胺基酸對於其抗菌能力的影響不大；另一方面，經由DTT還原分子內雙硫鍵的兩種重組蛋白質抗菌活性均有降低或喪失的現象，推論雙硫鍵的還原可能導致構形的改變而降低或失去抗菌活性。

關鍵字

Royalisin ； Royalisin-D ； 人造油體純化系統 ； 抗菌活性測試

並列摘要

Royalisin is a small antibacterial peptide isolated from royal jelly of the honeybee Apis mellifera. The molecular weight of royalisin is 5.5 kDa and it is stabilized by three intramolecular disulfide linkages, with six cysteine residues. Antibacterial peptides are key elements of insect innate immunity. The antimicrobial activity of royalisin against Gram-positive bacteria, Gram- negative bacteria and fungi has been known. Compared with another insect antibacterial peptide, royalisin have an extra stretch of 11 amino acid residues at the C-terminus. In this study, a recombinant protein, for which we proposed the name royalisin-D, was constructed without the 11 amino acid residues at the C-terminal of royalisin and was linked to the C-terminal of oleosin by an inteinS fragment. The recombinant protein was overexpressed in E.coli, purified by artificial oil body system and was subsequently released through self-splicing of inteinS induced by the changes of temperature. After harvested by concentrating the supernatant, we tested the activity of royalisin-D as a comparasion with royalisin via minimal inhibitory concentration assay (MIC), minimal bactericidal concentration assay (MBC), cell membrane permeability and MATS (microbial adhesion to solvents) methods. Furthermore, the recombianat proteins of royalisin and royalisin-D have also been treated with the reducing agent of disulfide bonds, dithiothreitol (DTT), to research the importance of our recombinant proteins. All our data shows that royalisin-D still had the antimicrobial activity for Staphylcoccus aureus, Staphylcoccus xylosus, Staphylcoccus intermedius B, Paenibacillus larvae and Pseudomonas aeruginosa. In our results, royalisin-D had similar antimicrobial activity to royalisin and the disulfide bonds are very important for antimicrobial activity. In other words, the disulfide bonds of royalisin are more important in its antimicrobial activity than the 11 amino acid residues at C-terminal.

並列關鍵字

Royalisin-D ； MIC assay ； MBC assay ； cell membrane permeability ； MATS

參考文獻

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