鮑氏不動桿菌 (Acinetobacter baumannii) 為嗜酸性之革蘭氏陰性絕對好氧菌，A. baumannii 為一伺機性的病原菌，且在寄主免疫能力變弱的情況容易發病，適應力強並廣泛存在於大自然環境中。本實驗室已分離多株對 A. baumannii 具溶裂性的噬菌體，其中以 ϕkm18p 具有最高的溶裂能力。在動物實驗中，ϕkm18p 可使受病菌感染的小鼠 100% 存活，因此本研究針對 ϕkm18p 溶菌素基因進行基因選殖，利用基因工程方式選殖出可表現溶菌活性之重組質體。
本研究將噬菌體ϕkm18p 的溶菌基因 orf693 選殖於 pET21b 構築成表現載體 pEorf693，轉殖於 E. coli C41 (DE3) 後誘導蛋白大量表現，得到分子量約為 70 kDa 的蛋白，但不具有溶菌活性。若純化蛋白不經變性，以 zymography 方式探討具活性的溶菌蛋白位置，結果溶菌區出現於 210 kDa 左右，因此推估，轉譯後的蛋白會於菌體中形成三聚體 (trimers) 結構。以 paper disc 進行溶菌分析，1μg 溶菌蛋白即有明顯溶菌活性，抑制圈會隨培養時間而擴大。在最小抑制濃度測試 (MIC) 中，添加 0.5 mg/ml 溶菌蛋白能抑制宿主細菌生長。

Acinetobacter baumannii is an acidiphilic, obligate, aerobic and gram-negative bacterium. It is an opportunistic pathogen and people will fall to ill when they were under weak immunity. We have isolated several lytic bacteriophages from the environment to lysis A. baumannii and the km18p has the highest lytic efficiency. Phage km18p provided 100% protection and survival rate of mice that were inoculated with A. baumannii KM18. So this study will focus on clonong the lysin gene and detecting the bacterial inhibition efficiency of the expressed proteins.
On this study, the lysin gene orf693 were cloned to pET21b to from the expression plasmid pEorf693. Plasmid pEorf693 was transferred to E. coli C43 (DE3) and induced by IPTG to over-expression. The induced protein showed the molecular weight about 70 kDa in SDS-PAGE, but it did not has lytic activity. If the purified proteins didn''t denatured and protein patterns with lysis activity were identified with zymogram method, we found that the protein pattern with 210 kDa molecular weight showed the lysis clear zone. We speculated that the translated lysin proteins were assembly as trimers structure in bacterium. When bacterium lysis activity was analysis with the paper disc, 1 μg/ml purified protein would present the obvious lysis activity and the diameter of inhibition zone would also increase during 120 hr. In the minimum inhibitory concentration (MIC) assay, purified proteins with minimum concentration of 0.5 mg/ml would inhibit the growth of A. baumannii KM18.

摘要 I
Abstract II
誌謝 III
目錄 IV
圖目錄 VI
表目錄 VII
壹、前言 1
貳、文獻回顧 3
一、Acinetobacter spp.之背景 3
二、A. baumannii 之抗藥性 4
三、噬菌體 lysin 蛋白簡介、抗菌機制及應用 5
A. Lysin 蛋白簡介 5
B. Lysin 抗菌機制 6
C. Lysin 之應用 7
四、Phage 及 Lysin治療之優點 8
参、研究目的 10
肆、材料與方法 11
【實驗材料】 11
一、菌種及噬菌體來源 11
二、基因選殖所使用之引子、宿主、質體及構築質體 11
三、儀器及藥品 11
四、培養基配製 11
五、藥品與緩衝溶液配製 12
【實驗方法】 12
一、菌種活化與菌株保存 12
二、噬菌體純化 12
三、噬菌體增殖 13
四、噬菌體濃縮與保存 13
五、噬菌體 DNA 抽取 14
六、質體 (plasmid) 抽取 14
七、重組質體製備 15
A. 聚合酶鏈鎖反應 (Polymerase Chain Reaction；PCR) 15
B. PCR 產物 Clean-up 16
C. 電泳法回收 DNA 片段 16
D. DNA 端點補齊 (Fill-in) 17
E. DNA 接合反應 (Ligation) 17
F. 限制酶切割 (Restriction Enzyme Digestion) DNA之分析 18
G.洋菜膠體電泳 (Agarose gel electrophoresis) 18
八、大腸桿菌勝任細胞 (Competent cells) 製備 19
九、勝任細胞轉型作用 (Transformation) 19
十、快速篩選重組質體 20
十一、重組質體蛋白表現 20
十二、重組蛋白濃縮及保存 21
十三、SDS-PAGE 蛋白質膠體電泳 21
A.膠體電泳 21
B.孔雀藍染色 (Coomassie blue) 22
C.Zymogram 方式分析蛋白活性 22
十四、Lysin 重組蛋白抑菌測試 23
A.紙錠測試 23
B.最小抑菌濃度測試 (Minimum inhibitory concentration；MIC) 23
十五、Lysin 重組蛋白耐溫測試 23
十六、蛋白質濃度檢測線 24
伍、實驗結果 25
一、Lysin 基因選殖 25
二、重組質體之蛋白質表現、純化與抑菌分析 25
三、溶菌蛋白之耐溫性測試 26
五、最小抑菌濃度測試 (Minimum inhibitory concentration；MIC) 27
陸、討論 28
柒、參考文獻 31
Appendix 2. 藥品與廠牌 48
Appendix 3. 培養基配方 49
Appendix 4. 藥品及緩衝溶液之配製 50
Appendix 5. ORF693 蛋白質之 DNA 序列 52

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