臺灣博碩士論文加值系統

中文摘要
本研究旨探討環境因子對腸出血性大腸桿菌 E. coli O157:H7 (enterohemorrhagic Escherichia coli O157:H7) 生長及產毒之影響，以及生產 E. coli O157:H7 單株抗體，並建立免疫-SPR 光譜分析系統。在 23 株 E. coli O157:H7 中，選出產毒能力最強的菌株 BCRC 13099 做為研究用菌株。溫度與 pH 值明顯影響 BCRC 13099 菌株之生長與產毒能力，在 15℃ 至 42.5℃，隨著溫度升高，菌株生長速度增加，但在 42.5℃ 與 37℃ 溫度生長速度相近，培養 6 小時菌數已經達 10 9 cfu/mL。菌株產毒能力亦隨溫度增加而增大，在 15℃、25℃、37℃ 及 42.5℃，所產 verotoxin 力價數分別為 252、2823、6132 與 18790。該菌在 pH 4.0 brain heart infusion broth (BHIB) 不生長，在 pH 10 BHIB 逐漸死亡，在 pH 5.0~pH 9.0 生長相近，但產毒能力完全不同。
以 BCRC 13099 菌體表面水溶性蛋白質萃取液免疫 BALB/c 小鼠，經五次免疫後血清中抗體含量為原始之 100 倍。取小鼠脾臟與骨髓瘤細胞 NS-1 進行融合。經 HAT 培養液篩選及限數稀釋，得到三株單株細胞 A7F-7B、G2H-4B 與 H9C-3D。抗體 G2H-4B 對 E. coli O157:H7 檢出率約為 95.7％ (22/23)，對 9 株非 E. coli 菌株之偽陽性比率為 0％。
抗體塗敷在 CM-5 晶片表面之最適濃度為 50 μg/mL。使用 Biacore 電漿共振儀，以塗敷抗體之 CM-5 晶片偵測 E. coli O157:H7 之菌體，檢出菌濃度為 107 cfu/mL，但若以破碎菌體進行偵測，檢出界限達 101~102 cfu/mL。

目錄
中文摘要.................................................Ⅰ
英文摘要.................................................Ⅲ
目錄.....................................................Ⅳ
圖目錄...................................................Ⅷ
表目錄...................................................Ⅹ
壹、前言..................................................1
貳、文獻整理..............................................3
2-1.1 大腸桿菌............................................3
2-1.2 病原性大腸桿菌......................................5
(1) 腸毒素性大腸桿菌 (Enterotoxigenic E. coli, ETEC)......5
(2) 腸病原性大腸桿菌 (Enteropathogenic E. coli, EPEC).....5
(3) 腸侵襲性大腸桿菌 (Enteroinvasive E. coli, EIEC).......6
(4) 腸出血性大腸桿菌 (Enterohemorrhagic E. coli, EHEC)....6
(5) 腸成簇性大腸桿菌 (Enteroaggregative E. coli, EAggEC...6
2-1.3 Escherichia coli O157:H7............................9
2-1.4 Escherichia coli O157:H7 之分離與鑑定..............11
(1) 培養基分離法.........................................11
A. Sorbitol-MacConkey agar 法............................11
B. 4-methylumbellifery-β-D-glucuronide 法................11
(2) 快速分析方...........................................12
A. 免疫分析法............................................12
Ⅰ. 酵素結合免疫分析法 (ELISA)...........................12
Ⅱ螢光免疫分析法 (Immunofluorescent filter technique)....12
Ⅲ.乳膠微粒凝集法 (Latex agglutination test).............12
Ⅳ.免疫轉印斑點法 (Petrifilm Test Kit-HEC)...............13
Ⅴ. 免疫色層分析法 (Singlepath GLISA Teststick)..........13
Ⅵ免疫磁珠分離法 (Immunomagnetic separation bead：IMS).....................................................14
B.分子生物學鑑定法.......................................14
Ⅰ. PCR法 (Polymerase chain reaction)....................14
Ⅱ. 核酸探針法 (DNA probe)...............................15
Ⅲ. DNA 雜交法...........................................15
2-2 單株抗體 (monoclonal antibody).......................16
2-3.1 表面電漿共振 (Surface plasmon resonance) 偵測......20
2-3.2 表面電漿共振於生物檢測上之應用.....................23
參、實驗設計.............................................24
肆、材料設備與方法.......................................25
4-1. 材料................................................25
4-1.1 菌株及細胞株.......................................25
4-1.2 實驗動物...........................................25
4-1.3 細菌培養基.........................................25
4-1.4 動物細胞培養.......................................25
4-1-5. 細胞致死之測定 (MTT assay)........................26
4-1.6 酵素連結免疫吸附分析法.............................27
4-1.7 單株抗體製作.......................................27
4-1.8 表面電漿共振 (surface plasmon resonance)...........27
4-2 儀器設備.............................................28
4-4 方法.................................................28
4-3.1 菌株保存、活化.....................................28
4-3.2 菌株生產 cytotoxin 能力測試........................29
4-3.3 菌體表面水溶性溶出物萃取...........................30
4-3.4 蛋白質定量.........................................31
4-3.5 單株抗體之生產.....................................31
4-3.6抗體固定至晶片......................................36
4-3.7 生物感測晶片最低偵測限制與專一性探討...............37
伍、結果與討論...........................................38
5-1 不同 E. coli O157:H7 產毒能力比較....................38
5-2不同溫度對 E. coli O157:H7 生長及產毒能力影響.........38
5-3不同 pH 對 E. coli O157:H7 生長及產毒能力影響.........40
5-4 E. coli O157:H7 單株抗體製備.........................41
5-4.1 小鼠免疫...........................................41
5-4.2 細胞融合與 HAT 之篩選..............................42
5-4.3 融合瘤細胞單株化...................................43
5-4.4 抗體專一性.........................................44
5-5 表面電漿共振.........................................45
5-5.1 抗體固定及最適濃度.................................45
5-5.2 不同濃度之全菌體對晶片表面抗體反應之 RU 值.........46
5-5.3不同濃度菌體破碎液對晶片表面抗體反應之 RU 值........48
5-5.4 晶片之專一性.......................................49
陸、結論.................................................52
柒、參考文獻.............................................53
圖目錄
圖一、E. coli O157:H7 BCRC 13099 在 BHIB 培養液中於 42.5℃、37℃、25℃ 與 15℃ 生長情形..............................61
圖二、E. coli O157:H7 BCRC 13099 在 BHIB 培養液中於 42.5℃、37℃、25℃ 與 15℃ 之毒素力價............................62
圖三、37℃ 培養之 E. coli O157:H7 BCRC 13099 在不同起始 pH 值之 BHIB 之生長情形 (A) 及 BHIB pH 值變化情形 (B).......63
圖四、37℃ 培養之 E. coli O157:H7 BCRC 13099 在不同起始 pH 值之 BHIB 之毒素力價.....................................64
圖五、經注射菌體表面萃取物後，小鼠血液中抗體力價變化.....65
圖六、以 HAT 培養液篩選融合細胞 (300 倍).................66
圖七、融合瘤限數稀釋後所形成的單一細胞株 (X40 倍)........67
圖八、抗體固定於 CM5 晶片之感應圖。抗 E. coli O157:H7 多株抗體溶於 pH 4.5 之醋酸緩衝溶液.............................68
圖九、以原子力顯微鏡觀察抗體固定後之晶片表面.............69
圖十、晶片固定不同抗體濃度後對 107 cfu/mL E. coli O157:H7 之感應圖...................................................70
圖十一、不同濃度之 E. coli O157:H7 全菌體感應圖..........71
圖十二、經二級抗體擴大反應後，不同濃度之 E. coli O157:H7 全菌體感應圖...............................................72
圖十三、以光學顯微鏡觀察注入E. coli O157:H7 (108 cfu/mL) 全菌後感測晶片表面.........................................73
圖十四、原子力顯微鏡觀察注入 E. coli O157:H7 全菌 (108 cfu/mL) 後之晶片表面.....................................74
圖十五、不同濃度之 E. coli O157:H7菌體破碎液之感應圖.....75
圖十六、經二級抗體擴大反應後，不同濃度之 E. coli O157:H7
菌體破碎液感應圖.........................................76
圖十七、以光學顯微鏡觀察注入E. coli O157:H7 菌體 (108 cfu/mL) 破碎液後之感測晶片表面...........................77
圖十八、原子力顯微鏡觀察注入 E. coli O157:H7 菌體 (108 cfu/mL) 破碎液後之晶片表面...............................78
圖十九、不同腸道科菌之感應圖 (107 cfu/mL)................79
表目錄
表一、不同 E. coli O157:H7 培養於 37℃ 之毒素力價........80
表二、以 ELISA 測定融合瘤細胞對 E. coli BCRC 13099 所產抗體.......................................................81
表三、不同單株抗體對不同 E. coli O157:H7 菌株 ELISA 反應之相對活性...................................................82
表四、不同單株抗體對非 O157:H7 型 E. coli 菌株及非 E. coli 菌株 ELISA 反應之相對活性................................83
表五、E. coli O157:H7 全菌在初次抗體及二次抗體之 RU 值變化.......................................................84
表六、E. coli O157:H7 菌體破碎液在初次抗體及二次抗體之 RU 值變化.....................................................85

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