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研究生:陳俐文
研究生(外文):Li-wen Chen
論文名稱:蛇毒心臟毒素抗菌作用機制
論文名稱(外文):Antibacterial Activity of Cardiotoxins from Naja naja atra and Naja nigricollis Venom
指導教授:張榮賢
指導教授(外文):Long-Sen Chang
學位類別:碩士
校院名稱:國立中山大學
系所名稱:生物醫學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2011
畢業學年度:99
語文別:中文
論文頁數:73
中文關鍵詞:抗菌作用心臟毒素脂多醣脂磷壁酸膜通透性
外文關鍵詞:cardiotoxinantibacterial actionlipopolysaccharidelipoteichoic acidmembrane permeability
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本論文主要探討台灣眼鏡蛇心臟毒素3 (CTX3) 和埃及金色眼鏡蛇心臟毒素gamma (toxin gamma) 對於細胞膜破壞活性與抗菌作用兩者之間的關係。 藉由抗菌實驗發現CTX3對於抑制金黃色葡萄球菌 (S. aureus,革蘭氏陽性菌) 生長具有優於抑制大腸桿菌 (E. coli,革蘭氏陰性菌) 生長的效果,而toxin gamma在大腸桿菌和金黃色葡萄球菌均具有相似程度的抑制生長能力,而利用propidium iodide (PI) 染劑發現隨著心臟毒素濃度增加均使細菌細胞膜滲透性增加之趨勢,於是進一步使用掃描式電子顯微鏡觀察發現CTX3和toxin gamma可造成細菌細胞膜不完整甚至破裂之形態。 由於革蘭氏陰性菌細胞壁含有脂多醣 (lipopolysaccharide,LPS), 而革蘭氏陽性菌富有脂磷壁酸 (lipoteichoic acid,LTA) 這類多醣類物質可保護細菌降低殺菌劑之作用,利用EDTA擾亂大腸桿菌的LPS結構以及抑制金黃色葡萄球菌的LTA生合成時,可增強CTX3和toxin gamma的抗菌效果,但是在競爭實驗中卻發現心臟毒素對LPS和LTA的結合性並無明顯差異。 根據先前研究,心臟毒素會形成類似通道的孔洞使脂質體內容物釋出,於是藉由人造細胞膜脂質體實驗發現CTX3對於金黃色葡萄球菌細胞膜組成具有較強的破膜活性,toxin gamma不論在模擬大腸桿菌或是金黃色葡萄球菌細胞膜組成之脂質體中均具有不錯的破膜效果,一旦加入LPS或LTA會抑制CTX3和toxin gamma破膜活性,提高CTX3和toxin gamma濃度可逆轉效果,因為LPS或LTA可抑制破膜活性,除此之外,當CTX3和toxin gamma在loop II的Met殘基被氧化時,確實會降低抗菌作用與破膜能力。 綜合以上結果,CTX3和toxin gamma以增加菌體細胞膜通透性為主要的抗菌作用機制。
The aim of the study is to investigate the causal relationship between membrane-damaging activity and antibacterial action of cardiotoxins from Naja naja atra (Taiwan cobra) cardiotoxin 3 (CTX3) and Naja nigricollis (Egyptian cobra) toxin gamma. Compared with that on Escherichia coli (E. coli, Gram-negative bacteria), CTX3 showed a greater growth inhibition on Staphylococcus aureus (S. aureus, Gram-positive bacteria). Antibacterial avtivity of toxin gamma toward E. coli and S. aureus was similar. Bactericidal action of cardiotoxins positively correlated with increase in membrane permeability of bacterial cells. Morphological examination showed that cardiotoxins disrupted the integrity of bacterial membrane. Cardiotoxins showed similar binding capability with lipopolysaccharide (LPS) and lipoteichoic acid (LTA), and destabilization of LPS layer and inhibition of LTA biosynthesis on cell wall promoted bactericidal effect of cardiotoxins on E. coli and S. aureus, respectively. CTX3 notably permeabilized model membrane of S. aureus and toxin gamma had similar activity on the permeabilization of bacterial model membrane used. Membrane-damaging activity of cardiotoxins was inhibited by either LPS or LTA, while increasing concentrations of cardiotoxins counteracted the inhibitory action of LPS and LTA. Oxidation of Met residues on loop II of cardiotoxins simultaneously attenuated membrane-permeabilizing activity and bactericidal effect of cardiotoxins. Taken together, our data indicate that antibacterial action of cardiotoxins depend on their ability to induce membrane permeability.
論文審定書 .................................................................................................................. i
致謝 ............................................................................................................................. ii
摘要 ............................................................................................................................ iii
Abstract ....................................................................................................................... iv
縮寫表 ..........................................................................................................................v
第一章、緒論 ..............................................................................................................1
1.1 蛇毒心臟毒素 .............................................................................................1
1.2 細菌細胞壁 .................................................................................................2
1.3 細菌細胞壁之致病機制 .............................................................................4
1.4 抗菌胜肽 .....................................................................................................4
1.5 抗菌胜肽之抗菌機制 .................................................................................6
1.6 脂質體簡介 .................................................................................................7
1.7 研究動機與目的 .........................................................................................8
第二章、實驗材料與方法 ..........................................................................................9
2.1 實驗材料 .....................................................................................................9
2.2 細菌培養基配方 .......................................................................................10
2.3 實驗方法 ...................................................................................................12
第三章、結果 ............................................................................................................18
3.1 蛇毒心臟毒素抗菌作用 ...........................................................................18
3.2 細菌細胞壁與抗菌作用之影響 ...............................................................20
3.3 以細菌細胞膜脂質體model 探討心臟毒素之破膜影響 .......................22
第四章、討論 ............................................................................................................24
圖表 ............................................................................................................................29
參考文獻 ....................................................................................................................48
附錄 ............................................................................................................................58
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