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研究生:李建輝
研究生(外文):Chien-Hui Li
論文名稱:霍亂弧菌多重藥物ABC轉運蛋白VcaM特性探討
論文名稱(外文):Characterization of multidrug ABC transporter VcaM from Vibrio cholerae
指導教授:林泓廷
指導教授(外文):Hong-Ting Lin
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:食品科學系
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:125
中文關鍵詞:ABC多重藥物轉運幫浦霍亂霍亂弧菌VcaM
外文關鍵詞:ABC multidrug efflux pumpCholeraVcaMVibrio cholerae
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霍亂 (Cholera) 一種由霍亂弧菌 (Vibrio cholerae) 引起的急性腹瀉疾病,會造成脫水、電解質流失。臨床上治療霍亂重症者所使用的抗生素多為norflexacin。本論文探討ABC (ATP-binding cassette) 轉運蛋白VcaM的特性,並期待將有助於對抗臨床上Vibrio cholerae日益增加的抗藥性。Pfam的二級結構針對VcaM蛋白質的序列分析發現序列中存在ABC轉運蛋白的序列特徵,其N端有6個transmembrane helices,而C端有nucleotide binding domain及其中的Walker A motif、Walker B motif及ABC signature。首先藉由培養帶有vcaM基因質體的E. coli C43 (DE3) 菌株,並於24℃下誘導表達VcaM蛋白6小時後收菌,將破菌獲得蛋白質液用親合性管柱 (nickel column) 純化並進行去鹽後,可由每公升E. coli培養液獲得1 mg的純化VcaM蛋白。以孔雀綠 (Malachite Green) 呈色分析法分析純化後VcaM蛋白活性,並由酵素動力學分析得出Vmax= 25.8 pmole/mg VcaM/min,Km= 368 μM,kcat= 0.03 S-1。在電泳及動態光散射 (Dynamic light scattering,DLS) 的結果發現VcaM蛋白 (溶於Triton X-100) 具有單、雙聚體。在最小抑菌濃度測試方面,本研究測試E. coli的藥物敏感菌株E. coli Kam3 (DE3) 及膜蛋白表現菌株E. coli C43 (DE3) 在表現VcaM蛋白的情況下,針對在Norflexacin、Tetracycline、Erythromycin及Kanamycin藥物毒性測試,發現E. coli C43 (DE3) 表現VcaM可增加Norfoxacin的抗性為控制組的4倍。
Cholera, as a result of Vibrio cholerae infection, causes diarrhea, dehydration and electrolyte imbalance. Antibiotics tetracycline and fluoroquinolones, such as norflexacin, were most frequently used to treat severe cholera patients. This thesis aimed to characterize multidrug ABC transporter VcaM from V. cholera, and it will help combat the increasing drug resistance of V. cholerae. By using the pfam database, analysis of VcaM protein sequence revealed its secondary structure where VcaM has a transmembrane domain (TMD) containing six transmembrane α-helices on the N-terminus and a nucleotide binding domain (NBD) containing motifs Walker A, Walker B and ABC signature on the C-terminus. E. coli C43 (DE3) cells harboring plasmid encoding VcaM protein were induced and the VcaM proteins were overexpressed for 6 hours at 24℃and purified by a nickel column and a desalting column, yielding of 1 mg VcaM per liter. ATPase activity tests of VcaM, analyzed by using malachite green assay, gave a Vmax of 25.8 pmole per mg VcaM per min, a Km of 368 μM and a kcat of 0.03 S-1. Gel electrophoresis and dynamic light scattering results indicated that Triton X-100 solubilized Vcam was a mixture of monomeric and dimeric proteins in solution. Inhibition test against antibiotics norflexacin, tetracycline, erythromycin and kanamycin indicated E. coli C43 overexpressing VcaM gave a rise of norflexacin antibiotic resistance for 4 fold.
誌謝 II
縮寫表 V
目錄 VII
圖目錄 XI
表目錄 XIII
壹、前言 1
貳、文獻整理 3
一、多重抗藥性 3
1.1 何謂多重抗藥性MDR 3
1.2 抗生素作用位置及機轉 8
二、多重藥物轉運蛋白 9
2.1 次級主動轉運蛋白 9
2.2 ABC轉運蛋白 9
2.2.1 何謂ABC轉運蛋白 10
2.2.2 ABC轉運蛋白變異造成的相關人類疾病 11
三、ABC轉運蛋白的結構與機制 12
3.1 高度保留的功能區域和特徵序列 12
3.2 ABC轉運蛋白的結構 14
3.3 核苷酸結合區的構形變化 15
3.4 轉運蛋白水解偶合的機制 16
四、格蘭氏陽性菌中ABC型的多重藥物抗性轉運蛋白系統 17
五、格蘭氏陰性菌中ABC型的多重藥物抗性轉運蛋白系統 18
六、V. cholerae多重耐藥機制 19
參、實驗設計 26
肆、實驗材料與方法 28
一、實驗材料 28
1. 資料庫及網路工具 28
2. 菌株與載體 28
2.1 vcaM基因來源 28
2.2 蛋白質表現用載體 28
2.3 E. coli菌株 29
2.4 Primers 29
3. 培養基與抗生素 30
3.1 培養基 30
3.2 抗生素 30
4. 標準品 30
4.1 磷酸根標準品配製藥品 30
4.2 孔雀綠 (Malachite Green) 呈色標準品配製藥品 30
5. 市售套組 30
6. 酵素與抗體 31
7. 化學藥品 31
8. 實驗設備 32
二、實驗方法 33
1. 遺傳操作 33
1.1 聚合酶鏈連鎖反應 33
1.2 DNA製備與電泳 34
1.3 限制酶剪切及DNA接合 34
1.4 轉形至E. coli 36
2. 蛋白質表達與純化 37
2.1 固定化金屬層析 37
2.2 離子交換層析 39
3. 蛋白質分析 39
3.1 電泳 39
3.2 蛋白質濃度測定 41
3.2.1 UV spectrophotometer 41
3.2.2 Bicinchoninic Acid (BCA) Assays 42
3.3 西方免疫轉漬法 42
4. 生化分析 44
4.1 藥物毒性測試 44
4.2 ATP酶活性測試 46
5. 生物物理分析 49
5.1 動態光散射 49
伍、結果與討論 50
一、VcaM蛋白序列分析 50
1.1 一般特性分析 50
1.2 功能區塊 (Domain) 與特徵序列 (Motif) 分析 50
二、vcaM基因cloning 52
2.1 PCR擴增vcaM基因 52
2.2 vcaM基因接合至pET21a載體 53
2.3 pET21a-vcaM construct轉形至E. coli 54
2.4 DNA sequencing 55
三、VcaM 蛋白表現及純化 55
3.1. VcaM蛋白親合性管柱純化 56
3.2 VcaM 蛋白SDS-PAGE與Western blot 56
3.3 VcaM蛋白層析管柱去鹽 (Desalting) 57
3.4 VcaM蛋白質定量分析 58
四、VcaM 蛋白之特性分析 58
4.1 動態光散射 (Dynamic light scattering, DLS) 59
4.2 VcaM蛋白ATP酶活性測試 60
4.3藥物毒性測試 (MIC, minimal inhibitory concentration) 64
柒、未來方向 69
捌、參考文獻 70

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