本論文主要目的在於研究牛血清蛋白(BSA)與白蛋白(OVA)經semicarbazide修飾羧基後對於蛋白質物化特性及抗菌活性的影響。質譜分析及RP-HPLC結果顯示修飾後的BSA與OVA分子量明顯增加，並且其親水性，CD spectra分析則顯示修飾羧基改變蛋白質二級結構。由抗菌實驗發現SEM-BSA可抑制革蘭氏陰性菌E. coli(大腸桿菌)與革蘭氏陽性菌S. aureus (金黃色葡萄球菌)的生長，但SEM-OVA僅可抑制S. aureus的生長，對E. coli則無明顯抗菌活性。降低E. coli細胞壁成分lipopolysaccharide (LPS)穩定度及抑制S. aureus合成lipoteichoic acid (LTA)均能提高SEM-BSA與SEM-OVA的抗菌活性。以propidium iodide (PI)染劑證明SEM-BSA及SEM-OVA可增加S. aureus細胞膜通透性；相對SEM-BSA，SEM-OVA對E. coli細胞膜通透性無明顯影響。使用脂質體模擬細菌細胞膜，探討SEM-BSA及SEM-OVA與細菌細胞膜組成脂質體的交互作用，證明SEM-BSA與SEM-OVA可引發模擬S. aureus細胞膜脂質體膜融合且具有破膜活性；與相對SEM-BSA不同，SEM-OVA對E. coli細胞膜組成脂質體則不具破膜及膜融合活性，同時亦發現LPS與LTA可抑制SEM-BSA及SEM-OVA的破膜作用。SEM-BSA對模擬細菌細胞膜脂質體具較高親合性，並可插入細胞膜內，破壞細胞膜結構；SEM-OVA對細菌細胞膜脂質體親合性較低， 且SEM-OVA與E. coli及S. aureus脂質體膜結合方式不同。綜合上述實驗結果顯示SEM-BSA與SEM-OVA可透過破壞細菌膜而產生抗菌效果，而SEM-OVA與E. coli細胞膜結合方式可能使其無法有效對E. coli產生抗菌活性。

The aim of this study is to investigate the physicochemical properties and antimicrobial activities of semicarbazide-modified bovine serum albumin (BSA) and ovalbumin (OVA). MALDI-TOF and RP-HPLC showed that modification of carboxyl groups caused an increase in molecular weight and hydrophilicity of SEM-BSA and SEM-OVA compared with those of BSA and OVA. CD spectra measurement revealed that modification of carboxyl groups caused a change in the secondary structure of BSA and OVA. SEM-BSA exhibited a growth inhibition on E. coli and S. aureus, while SEM-OVA only shows bactericidal activity against S. aureus. Destabilization of structural stability of lipopolysaccharide (LPS) or inhibition of lipoteichoic acid (LTA) synthesis promoted antibacterial activity of SEM-BSA and SEM-OVA. Propidium iodide (PI) staining indicated that SEM-BSA and SEM-OVA induced membrane permeability of S. aureus. Compared to SEM-BSA, SEM-OVA insignificantly affected the membrane permeability of E. coli. SEM-BSA and SEM-OVA induced membrane fusion and permeability of S. aureus membrane-mimicking vesicles. Unlike SEM-BSA, SEM-OVA did not damage E. coli membrane-mimicking vesicles. Both LPS and LTA suppressed membrane-damaging activity of SEM-BSA and SEM-OVA. SEM-BSA showed higher binding affinity with bacterial membrane-mimicking vesicles compare to SEM-OVA. Moreover, SEM-BSA penetrated into membrane and perturbed the membrane structure. The interacted-mode of SEM-OVA with S. aureus and E. coli membrane-mimicking vesicle differed. Taken together, the antibacterial action of SEM-BSA and SEM-OVA are related to their ability to damage bacterial membrane. The membrane-interacted mode of SEM-OVA may elucidate its inability to display antibacterial activity against E. coli.

論文審定書 i
論文公開授權書 ii
致謝 iii
摘要 iv
Abstract v
目次 vi
圖次 vii
表次 x
英文縮寫表 xi
第一章、緒論 1
1.1 傳統抗生素的抗菌機制與瓶頸 1
1.2 細菌細胞壁組成與致病機 1
1.3 抗菌胜肽與抗菌蛋白 2
1.4修飾蛋白質使其具有抗菌活性 3
1.5 BSA與OVA具有與脂質膜交互作用的潛力 3
1.6 研究目的 4
第二章、實驗材料與方法 6
2.1實驗材料 6
2.2細菌培養配方 8
2.3實驗方法 9
2.3.1製備羧基修飾蛋白 9
2.3.2 MALDI-TOF質譜分析 9
2.3.3 Circular dichroism (CD，Jasco J-810 spectropolarimeter)光譜分析 9
2.3.4逆向高效能液相層析法 (RP-HPLC) 9
2.3.5 Bacterial strains 9
2.3.6 抗菌能力分析 9
2.3.7 測試細菌細胞膜通透性 10
2.3.8 製備脂質體 10
2.3.9 Calcein螢光釋放分析 11
2.3.10 膜融合能力分析 12
2.3.11 FPE螢光分析 12
2.3.12 Polydiacetylene (PDA)呈色反應 13
2.3.13 以掃描式電子顯微鏡觀察處理修飾蛋白後的細胞型態變化 13
2.3.14 分析修飾蛋白和plasmid DNA親和力 13
第三章、結果 15
3.1羧基修飾蛋白物化特性分析 15
3.2 探討SEM-BSA與SEM-OVA的抗菌能力 15
3.3干擾細胞壁合成及穩定性對抗菌能力的影響 15
3.4 處理修飾蛋白和細菌細胞膜通透性改變之關連性 16
3.5 以脂質體探討SEM-BSA與SEM-OVA的破膜特性 16
3.6 探討以SEM-BSA及SEM-OVA處理脂質體對於膜融合及穩定性的影響 17
3.7 比較SEM-BSA和SEM-OVA對模擬細菌脂質體的親和性 17
3.8 處理修飾蛋白造成細菌表面型態改變 17
3.9 SEM-BSA及SEM-OVA可和plasmid DNA結合 18
第四章、討論 19
附表 23
附圖 25
參考文獻 40
會議發表著作 48
附錄 49

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