臺灣博碩士論文加值系統

霍亂是由霍亂弧菌所造成的傳染疾病，常經由糞便或汙染的水源傳染，病患會有急性且嚴重的脫水現象，主要流行在亞洲及非洲，目前主要針對霍亂主要透過抗生素進行治療，然而隨著具有抗藥性的菌株出現，使用抗生素進行治療已越來越困難。先前研究發現霍亂弧菌中有一 ABC 轉運蛋白 VcaM，其被證實可轉運多種抗生素，而本研究指出 VcaM 亦具有轉運巨環類藥物的能力，且其轉運的方式不須透過次級轉運蛋白的協助。將 VcaM 表現於 E. coli Kam3 菌株中發現可使細胞增加巨環類藥物的抗性，erythromycin、clarithromycin、azithromycin、spiramycin 分別提升 8 倍、4 倍、4 倍 和 16 倍，若將 VcaM 表現於 E. coli TG1 (ΔtolC) 中，僅能提升 2 倍、0.5 倍、1 倍 和 0.125 倍。而在 H33342 累積試驗中，在 Kam3 菌株表現 VcaM 可大幅提升細胞轉運 H33342 的能力，在 TG1 菌株中則無法進行 H33342 的轉運，顯示 VcaM 為 TolC 依賴型的轉運蛋白，而在累積試驗中添加次級轉運蛋白抑制劑 PAβN 發現對於 VcaM 的轉運能力並無顯著影響，故推測 VcaM 轉運基質的方式應為透過膜融合蛋白和 TolC 之間產生連接，而非透過次級轉運蛋白將基質排出細胞。

Cholera is an infectious disease caused by Vibrio cholerae, mainly via infected water and fecal, causing acute and severe dehydration to the patients, mainly in Asia and Africa. At present, the main treatment for cholera through antibiotics, but it has become increasingly difficult to deal with the multidrug resistance strains. Previous studies have revealed that chromosomally encoded ABC transporter protein VcaM in Vibrio cholerae could confer resistance to various antibiotics. In this study, we indicated that E. coli overexpressing VcaM was able to confer resistance against macrolides in the absence of secondary active transport proteins. Drug sensitive E. coli strain Kam3 overexpressing VcaM could increase the resistance to macrocyclic drugs by 4 to 16 folds, but the resistance was abolished in a TolC-deletion (an outer membrane protein) E . coli strain TG1, indicating the resistance is TolC dependent. A similar result was observed in the accumulation test, where the reduction of H33342 accumulation in the cells were observed in Kam3, but not in TG1, overexpressing VcaM. Moreover, the addition of PAβN, a general secondary active transporter inhibitor, to Kam3 overexpressing VcaM did not cause any significant effect on drug resistance and dye accumulation. Thus, we proposed that VcaM confers a TolC-depedent drug resistance against macrolides in the absence of secondary active transporters.

摘要 II
Abstract III
目錄 IV
表、圖目錄 IX
縮寫表 X
壹、前言 1
貳、文獻回顧 3
2.1. 認識抗生素 & 抗生素抗藥性 3
2.1.1. 抗生素的發現 3
2.1.2. 抗生素的種類及作用機制 4
2.1.3. 抗生素抗藥性 5
2.1.4. 多重抗藥性 6
2.1.5. 細菌抗藥性機制 7
2.2. 多重藥物轉運蛋白 9
2.2.1. 外排轉運蛋白 9
2.2.2. 轉運蛋白分類 9
2.2.3. ABC 主動轉運幫浦 (ATP-binding cassette transporters) 10
2.2.4. VcaM 12
2.3.1. 霍亂 13
2.3.2. 霍亂弧菌 14
2.3.3. 霍亂毒素 16
2.3.4. 霍亂的臨床症狀&治療方式 16
2.4. 基因敲除 (knock-out) 17
2.4.1. 同源重組 17
2.4.2. 使用環狀質體進行基因敲除 18
2.4.3. 基因剔除的意義 19
參、實驗設計 21
肆、實驗材料與方法 23
4.1. 實驗材料 23
4.1.1. 載體與菌株 23
4.1.1.1. VcaM 來源 23
4.1.1.2. 菌株與載體 23
4.1.2. Primers 24
4.1.3. 培養基 25
4.1.4. 抗生素 25
4.1.5. 化學藥品 26
4.1.6. 市售套組 26
4.1.7. 酵素與抗體 26
4.1.8. 標準品 28
4.1.9. 染劑 28
4.1.10. 實驗儀器與耗材 28
4.1.11. 管柱 29
4.1.12. 反應緩衝液 29
4.1.12.1. PCR reaction buffer 29
4.1.12.2. 6X DNA loading buffer 30
4.1.12.3. 50X TAE buffer 30
4.1.12.4. Restriction enzyme reaction buffer 30
4.1.12.5. Ligase reaction buffer 31
4.1.12.6. TFB1 buffer 31
4.1.12.7. TFB2 buffer 31
4.1.12.9. A buffer 32
4.1.12.10. B buffer 32
4.1.12.11. Running gel (8%) 32
4.1.12.12. Stacking (4%) 33
4.1.12.13. 10X MOPS running buffer 33
4.1.12.14. Coomassie Brilliant Blue G-250 stain solution 33
4.1.12.15. Transfer buffer 33
4.1.12.16. Wash buffer 34
4.1.12.17. Phosphate stock 34
4.1.12.18. Blocking buffer 34
4.1.12.19. PBS buffer 34
4.2. 實驗方法 36
4.2.1. 建構基因敲除載體 36
4.2.1.1. T4 多聚核苷酸激酶反應 36
4.2.1.2. 聚合酶鏈鎖反應 36
4.2.1.3. DNA 電泳與純化 37
4.2.1.4. TA clone 37
4.2.1.5. 限制酶剪切與 DNA 接合作用 37
4.2.1.6. 重組質體的轉型作用 39
4.2.1.7. 基因敲除菌株製備 40
4.2.2. 蛋白質表現與純化 40
4.2.2.1. 蛋白質表現 40
4.2.2.2. 蛋白質純化 41
4.2.3. 蛋白質特性分析 41
4.2.3.1. 蛋白質電泳 41
4.2.3.2. 西方點墨法 (Western blot) 42
4.2.3.3. VcaM 巨環內脂類藥物轉運測試 43
4.2.3.4. 藥物累積測試 43
伍、結果與討論 45
5.1. VcaM 於大腸桿菌中之藥物抗性測試 45
5.1.1. 在大腸桿菌 Kam3 中表現 VcaM 可提高 E. coli 對於巨環類藥物的抗性 45
5.1.2. 在大腸桿菌 Kam3 中表現 VcaM 可提高 E. coli 轉運 H33342 的能力 46
5.1.3. 使用 ATPase 抑制劑可降低 VcaM 轉運 H33342 的能力 46
5.2. VcaM 在大腸桿菌中的外排方式 48
5.2.1. 在大腸桿菌 TG1 中表現 VcaM 並無法提高 E. coli 對於巨環類藥物的抗性 48
5.2.2. 在大腸桿菌 TG1 中表現 VcaM 無法提升轉運 H33342 的能力 48
5.2.3. 添加次級轉運蛋白抑制劑 VcaM 無法提升轉運 H33342 的能力 49
5.3. 基因敲除載體建構 51
陸、結論 52
柒、參考文獻 53

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