臺灣博碩士論文加值系統

大腸桿菌素是去氧核醣核酸型式的細菌毒素。當大腸桿菌遭遇環境壓力時會分泌此毒素殺害其它細菌以增加其在此環境中的競爭力。當大腸桿菌素在細胞中被轉譯時，會同時產生一免疫蛋白質與毒性區域結合以中和其毒性因而避免此毒素對菌體自身所造成傷害。經轉譯後，大腸桿菌素與免疫蛋白質會以異體複合物的形式分泌到細胞外。在目前已知大腸桿菌素與免疫蛋白質的解離平衡常數約為10-14至10-17。因此在大腸桿菌素入侵敏感細胞的過程中如何與免疫蛋白質分離?大腸桿菌素如何單獨進入敏感細胞中釋放其細胞毒性?這等都是懸而未決和是一個值得研究的課題。
在本篇報告中發現，只有大腸桿菌素中的毒性區域才能進入菌體中，顯示此毒素在進入細胞的過程中是經過蛋白質切割的過程。進一步發現在胞膜區間(periplasmic space)中有部份蛋白質會專一性的作用在大腸桿菌素上。經由蛋白質定序與電腦比對發現這些會與大腸桿菌素作用的蛋白質包括：有glucose-1-phosphatase，maltose binding protein E與flagellin等，所以此等蛋白質可能有參與桿菌素在胞膜中位移(translcoation)的現象。此外，經由一些實驗也發現免疫蛋白會在胞膜區間中堆積，因此我們認為免疫蛋白質會在胞膜區間中與大腸桿菌素分離造成了免疫蛋白堆積的現像。
總合以上的結果，我們對於去氧核醣核酸型式的大腸桿菌素進入菌體中的移位過程提出了一個模型。也就是大腸桿菌素與免疫蛋白複合物在與敏感細胞結合後，在胞膜區間中的某些蛋白質會將大腸桿菌素的C端毒性區域切割，因而促使C端的毒性區域能與免疫蛋白質分離。此外，由於胞膜區間有較低的pH值，造成毒性區域結構上的些微變化，這便是使得免疫蛋白質得以分離的其中一種原因。在胞膜間區分離後的毒性區域，再經過未知的機制穿越內膜而進入細胞中以執行其細胞毒性。

Colicins of DNase type are bacteriocin which released from E. coli during environmental stress. These proteins are inactivated upon the binding of immunity protein to its toxicity domain. Recent study indicated a high affinity binding (Kd = 10-14 — 10-17 M) between colicins and their immunity proteins (Col-Imm). These complexes, under normal conditions, do not dissociate easily. However, only colicins dissociated from Col-Imm complex which enter and kill the target cells. The mechanism attributes to dissociation of Col-Imm complex prior to enter the cytoplasm remains unknown. Our recent studies revealed that colicin must be cleaved prior to launch its cytotoxic effect and only the fragment containing toxicity domain can be translocated into target cell. Certain proteins in the periplasmic space bind specifically to colicin. Protein sequencing results showed that proteins in the periplasmic region specifically interact with ColE7 include glucose-1-phosphatase, maltose binding protein, flagellin, indicating that these proteins may play an important role for translocating colicin through the membrane. Since immunity protein is accumulated in the periplasmic space, it''s accumulation may be attributed to the dissociation of ColE7-Imm complex in the periplasmic space. Thus, a possible mechanism for colicin is proposed. After the binding of ColE7-Imm complex to its target cell, toxicity domain in the C terminal of colicin will be cleaved by certain periplasmic proteins leading to the dissociation of Imm protein with the toxicity domain in periplasmic space. Moreover, the low pH condition in periplasmic space might play a role for the dissociation of toxicity domain and immunity protein. Eventually, the processed toxicity domain translocates into cytoplasm through the inner membrane by an unknown mechanism to launch its cytotoxic effect in sensitive cells.
英文摘要…………………………………………….……………………Ⅲ
壹、 序論…………………………………………………………….1 ~ 12
一、背景介紹………………………………………………………1
(一)大腸桿菌素ColE7基因與其操縱子上其它基因的功能……..1
(二)大腸桿菌素ColE7基因與其操縱子的調控…………………..2
(三)大腸桿菌素的分類……………………………………………..3
(四)大腸桿菌素的作用機制………………………………………..4
二、大腸桿菌素與免疫蛋白之酵素動力學研究概況……………….…4
(一)大腸桿菌素與免疫蛋白之平衡分解常數研究………………..5
(二)大腸桿菌素及免疫蛋白質之間的作用力研究………………..5
三、大腸桿菌素進入敏感細胞之途徑……………………………….…6
(一)Tol系統中各個蛋白質的功能…………………………………7
(二)大腸桿菌素向細胞內運輸的過程……………………….…….8
四、大腸桿菌素運輸至細胞體外之途徑………………………….……9
五、大腸桿菌素的重要性…………………………………….…….…..10
六、實驗目的………………………………………………….………...11
貳、 實驗材料與方法……………………………………………..13 ~ 46
一、 細菌菌株……………………………………………………..…… 13
二、 質體及其建構………………………………………………..…… 13
三、 化學藥品與酵素……………………………………………..…… 14
四、 一般研究的方法…………………………………………….. 15 ~ 26
(一)細菌培養……………………………………………….……... 15
(二)在大腸桿菌中質體DNA之分離與純化……………..…….... 16
(三)大腸桿菌中全部DNA (total genomic DNA) 之分離與純化..18
(四)聚合鏈鎖反應……………………………………………... 19
(五)限制的切割……………………………………………..…. 20
(六)CIAP處理……………………………………………………..20
(七)Klenow反應處理……………………………………………...21
(八)DNA的連接反應………………………………………..…… 21
(九)洋菜膠體電泳……………………………...…………………. 22
(十)從洋菜膠中回收DNA片段及純化…………...……………... 23
(十一)細菌的轉形作用……………………………………...……. 24
(十二)選殖菌株之篩選-細菌質體之快速篩選法……………...… 26
五、一般蛋白質之純化……………………………..……………… 26 ~ 39
(一)大腸桿菌素與免疫蛋白之純化………………………………. 26
(二)大腸桿菌胞膜區間蛋白質之純化與分類分離………....……. 30
(三)蛋白質含量之測定……………………………………………..33
(四)蛋白質電泳……………………………………………………..34
(五)蛋白質染色……………………………………………………..38
六、胞膜間區蛋白質與大腸桿菌素互動之研究……………………39 ~ 41
(一)分子篩管柱……………………………………………………..39
(二)生物感應器……………………………………………………..39
七、西方墨點測試………………………………………………………...41
八、遠西墨點測試………………………………………………………...42
九、N端蛋白質序列分析…………….…………………………………...43
十、進入菌體內大腸桿菌素片段的純化………………………………..44
十一、胞膜間區蛋白質與大腸桿菌素作用之實驗方法………….…….44
十二、菌體外與菌體內免疫蛋白的純化………………………….44 ~ 46
(一)在上清液中免疫蛋白的純化…………………………….……44
(二)在菌體內免疫蛋白的純化…………………………………….45
(三)在胞膜間區中免疫蛋白的純化……………………………….46
參、 結果…………………………………………….…..……. 47 ~ 57
一、大腸桿菌素ColE7經胞膜位移進入細菌內最終產物的探討…… 47
二、胞膜間區蛋白質與大腸桿菌素ColE7互動之研究………… 48 ~ 52
(一)胞膜間區蛋白質與大腸桿菌素ColE7的作用環境…………..49
(二)胞膜間區蛋白質分類分離物與大腸桿菌素ColE7作用之研究……………………………………………………………....52
三、胞膜間區中能與大腸桿菌素ColE7作交互作用之蛋白質的鑑定與純化………………………………………………………………….53
四、大腸桿菌素ColE7內金屬離子對於胞膜間區蛋白質之影響…….55
五、免疫蛋白與大腸桿菌素ColE7分離後所在的位置………….56 ~ 58
(一)在細胞外免疫蛋白存在的情況………………………………..57
(二)在細胞內免疫蛋白存在的情況………………………………..57
(三)在細胞間區免疫蛋白存在的情況……………………………..57
肆、 討論……………………………………………………….. 59 ~ 57
一、大腸桿菌素ColE7進入細胞的形式…………………………...…. 60
二、胞膜間區蛋白質對於大腸桿菌素ColE7在胞膜位移過程中的影響……………………………………………………………….61 ~ 62
(一)胞膜間區蛋白質與大腸桿菌素ColE7專一性的作用………..62
(二)胞膜間區蛋白質與大腸桿菌素ColE7作用環境之探討……...63
三、胞膜間區蛋白質分類分離物與大腸桿菌素ColE7作用情況之研究…………………………………………………………………….64
四、與大腸桿菌素ColE7作用之胞膜間區蛋白質……………….64 ~ 66
(一) Glucose-1-phosphatase的基本性質與可能功能………...……65
(二) Maltose binding protein E的基本性質與可能功能……...……66
(三) Flagellin的基本性質與可能功能…………………………..…66
(四)Outer membrane porin F的基本性質與可能功能……………..67
五、大腸桿菌素ColE7內金屬離子與免疫蛋白在大腸桿菌素運輸中所扮演的色…………………………………………………..……68~ 71
(一)大腸桿菌素ColE7內金屬離子對於此蛋白質運輸的影響…..69
(二)免疫蛋白與大腸桿菌素ColE7分離後所在之位置…………..70
六、大腸桿菌素與免疫蛋白複合物進入菌體內的位移模型………….72
伍、圖表…………………………………………………………… 73 ~ 114
陸、參考文獻……………………………………………………115 ~ 123

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