臺灣博碩士論文加值系統

Verotoxin（VT）為腸出血性大腸桿菌（enterohemorrhagic E.coli, EHEC）主要毒力因子之一，可造成人類血痢和溶血性尿毒症候群 （hemolytic uremic syndrome, HUS），而在動物方面可造成動物的下痢，嚴重時甚至造成死亡，是一種人畜共通傳染疾病。VT依照胺基酸序列相異程度的不同可分成VT-I及VT-II兩群，不論是VT-I或VT-II在蛋白質結構上又可分成A及B兩種次單位，且兩者在A和B次單位之相似度為55％及57％。VT致病機制為B次單位會與宿主細胞受器結合，使得VT藉胞吞作用進入宿主細胞，導致了細胞的死亡。本論文之目的在於找出VT B次單位具抗原性區域，以供未來開發疫苗之用。實驗首先利用蛋白質分析軟體分析及預測可能具抗原性並且為高度保留區之胺基酸區段，再設計引子以聚合酶連鎖反應（polymerase chain reaction, PCR）增幅出VT-I B次單位之VT-I-1、VT-I-2與VT-II B次單位之VT-II-1、VT-II-2四段具抗原性的片段基因，分別以pET32a表現載體進行選殖，將構築好之載體轉形入BL21（DE3）pLysS中，以IPTG誘導蛋白質表現。接著利用金屬螯合層析法（metal chelate affinity chromatography）將表現之重組蛋白進行純化，並將純化後之四段重組蛋白分別免疫BALB/c鼷鼠四次，以生產四種不同的多株抗體。將這些抗體利用西方轉漬（Western blotting）分析上述重組蛋白，結果顯示此四種多株抗體皆可與上述4個重組蛋白反應產生呈色條帶，另無插入基因之pET32a重組蛋白也有條帶的產生，推測為pET32a融合蛋白所產生之抗體引發的免疫反應，而每個重組蛋白都有此融合蛋白，於是造成西方轉漬分析實驗呈現偽陽性反應。為解決此問題，本論文另以一不同於pET32a融合蛋白之pGEX 5X-3表現載體，表達VT-I及VT-II B次單位之全長蛋白，並以此全長重組蛋白作為抗原，重新進行西方轉漬分析。結果顯示四種以pET32a表達之重組缺損蛋白所誘導的鼷鼠抗體，皆會與重組VT-I及VT-II B次單位全長蛋白產生反應，但是並不會與無插入基因之pGEX 5X-3表現重組蛋白產生反應。因此，表達之缺損蛋白確含有VT B次單位之抗原決定位（epitope），且本實驗利用兩種不同融合蛋白表達系統可有效解決因抗融合蛋白抗體所產生之免疫交叉反應。為了解重組蛋白誘導之鼷鼠抗體是否具中和VT能力，於是進行VT毒性中和試驗。首先利用polymyxin B分別萃取出EHEC分泌之VT-I及VT-II外毒素，以Vero cells測試其是否可引起細胞病變並算出其50%之毒性劑量（50% cytotoxic dose, CD50）。再將經連續稀釋之四種鼷鼠抗體與混合4倍量CD50之外毒素混合，置於室溫下感作1小時。加入Vero cells，放入培養箱中培養3天，在置於光學顯微鏡下觀察細胞型態有無發生變化。結果顯示，2個VT-I重組缺損蛋白中，以VT-I-2具中和毒性能力，毒性中和力價為80倍；而VT-II之2個重組缺損蛋白VT-II-1及VT-II-2則皆具中和毒性能力，毒性中和力價皆為80倍。由此得知本實驗結構所生產之四段重組缺損蛋白皆可誘導鼷鼠產生對抗VT的抗體，但以VT-I-2、VT-II-1及VT-II-2具較佳之中和毒性能力，可供進一步測試混合重組蛋白作免疫元誘導中和抗體能力，以利於未來發展次單位疫苗之用。

Verotoxin（VT）is a major virulent factor of enterohemorrhagic E.coli（EHEC）leading to diarrhea and hemolytic uremic syndrome（HUS）in human. It can also cause diarrhea in animals and even death in some serious cases. VT is classified as VT-I and VT-II base on amino acid sequences. All VT-I and VT-II contain subunit A and B which have 55-57% homology in their amino acid sequences. The B subunit of VT-I binds to its host receptor and then this complex is engulfed by endocytosis leading to death of the cells. The purpose of this study is to identify the antigenic regions of subunit B of VT for development of subunit vaccines. The amino acid sequences with high antigenicity were predicted and amplified by PCR. The correct amplicons were cloned into pET32a vectors, transformed into competent cells, and expressed after induction with IPTG. BALB/c mouse were immunized with the expressed proteins purified with HiTrap affinity columns. Sera from immunized mice were used to probe the recombinant proteins in Western blots. However, results indicated sera reacted with all expressed proteins even negative controls. This suggested these antibodies were induced by fusion tags in pET32a vectors were used for expression of subunit B of VT. Results indicated sera from immunized mice specifically reacted with recombinant proteins. Therefore, epitopes of B subunit of VT were included in the recombinant proteins. Exotoxins of EHEC extracted by polymyxin B were added into Vero cells to calculate 50% cytotoxic dose based on cytopathic effects（CPE）. All sera from immunized mice were mixed with dosages of four CD50 of exotoxins to understand their neutralization titers. Results showed sera raised against VT-I-2, VT-II-1, and VT-II-2 proteins exhibited strong neutralization capabilities with titers up to 80 folds. These results offered useful information for further development of subunit vaccines.

目 錄
中文摘要........................................................................................................ I

Abstract.......................................................................................................... III

誌謝................................................................................................................ V

目錄................................................................................................................ VI

圖表目錄........................................................................................................ IX

第1章 前言.................................................................................................. 1
第2章 文獻回顧.......................................................................................... 3
2. 1 大腸桿菌感染宿主之策略................................................................. 3
2. 2 致病性大腸桿菌................................................................................. 4
2. 3 腸出血性大腸桿菌之起源................................................................. 5
2. 4 腸出血性大腸桿菌之致病因子......................................................... 5
2. 4. 1 Verotoxin（VT）........................................................................ 5
2. 4. 2 腸附著因子（Intestinal adherence factors）................................ 7
2. 4. 3 Enterohemolysin.......................................................................... 8
2. 5 EHEC之感染症狀.............................................................................. 9
2. 6 EHEC之傳染途徑.............................................................................. 10
2. 7 EHEC之檢測方法.............................................................................. 10
2. 8 腸出血性大腸桿菌感染症之治療與疫苗發展................................. 12
第3章 材料與方法...................................................................................... 14
3. 1 實驗材料............................................................................................. 14
3. 1. 1 菌株來源...................................................................................... 14
3. 1.1.1 EHEC來源.............................................................................. 14
3. 1.1.2保存質體及表現用宿主......................................................... 14
3. 2 實驗方法............................................................................................. 14
3. 2. 1 出血性大腸桿菌VT抗原性基因之選殖與表現....................... 14
3. 2.2.1引子設計................................................................................. 14
3. 2. 2 核酸萃取...................................................................................... 15
3. 2. 3 出血性大腸桿菌VT抗原性基因之選殖................................... 16
3. 2.3.1聚合酶鏈鎖反應..................................................................... 16
3. 2.3.2電泳分析................................................................................. 16
3. 2.3.3 PCR產物純化........................................................................ 16
3. 2.3.4接合作用................................................................................. 17
3. 2.3.5轉形作用................................................................................. 17
3. 2.3.6菌落之篩選............................................................................. 17
3. 2.3.7小量質體DNA之萃取與純化............................................... 17
3. 2.3.8限制酶切割確認重組之質體DNA........................................ 18
3. 2.3.9細菌之保存............................................................................. 18
3. 2. 4 出血性大腸桿菌VT抗原性基因之表現................................... 18
3. 2.4.1表現載體與基因片段之處理................................................. 18
3. 2.4.2接合作用................................................................................. 18
3. 2.4.3轉形作用................................................................................. 19
3. 2.4.4菌落篩選................................................................................. 19
3. 2.4.5質體純化................................................................................. 19
3. 2.4.6限制酶切割確認重組成功與否............................................. 19
3. 2.4.7蛋白質表現............................................................................. 19
3. 3 菌體蛋白質之抽取............................................................................. 20
3. 4 SDS-PAGE蛋白質電泳...................................................................... 20
3. 5 蛋白質之純化..................................................................................... 20
3. 6 蛋白質的濃縮與透析......................................................................... 21
3. 7 蛋白質之定量..................................................................................... 21
3. 8 多株抗體之製備................................................................................. 22
3. 9 西方轉漬分析（Western blotting assay）......................................... 22
3. 10腸出血性大腸桿菌之外毒素萃取..................................................... 23
3. 11外毒素之毒性測試............................................................................. 23
3. 12毒素中和測試..................................................................................... 24
第4章 結果與討論...................................................................................... 25
4. 1 腸出血性大腸桿菌VT抗原性基因之分析比對............................... 25
4. 1. 1 VT-I-1、VT-I-2、VT-II-1、VT-II-2基因選殖及定序.................. 25
4. 1. 2 胺基酸序列比對.......................................................................... 25
4. 2 VT-I-1、VT-I-2、VT-II-1、VT-II-2蛋白質表現................................... 32
4. 3 收集不同誘導時間之四段基因表現蛋白......................................... 32
4. 4 蛋白質純化......................................................................................... 32
4. 5 VT-I B及VT-II B之選殖與表現...................................................... 38
4. 5. 1 VT-I B及VT-II B之選殖.......................................................... 38
4. 5. 2 重組蛋白VT-I B及VT-II B之表現......................................... 39
4. 6 西方轉漬分析..................................................................................... 39
4. 6. 1 重組蛋白VT-I-1、VT-I-2、VT-II-1、VT-II-2之 西方轉漬分析.............................................................................. 39
4. 6. 2 重組蛋白VT-I B及VT-II B之西方轉漬分析........................... 40
4. 7 毒性測試............................................................................................. 47
4. 8 中和毒性測試..................................................................................... 47
第5章 結論.................................................................................................. 49
參考文獻........................................................................................................ 53
附錄................................................................................................................ 64
作者簡介........................................................................................................ 72
圖表目錄
圖 1、 6種致病性大腸桿菌獨特辨識宿主黏膜細胞之策略.................... 3
圖 2、 Shiga toxin進入細胞質路徑圖，VT也是以相同路 徑進入細胞質................................................................................... 8
圖 3、 EHEC之致病因子基因位置............................................................ 9
圖 4、 腸出血性大腸桿菌Verotoxin之VT-I-1、VT-I-2、 VT-II-1、VT-II-2之PCR電泳圖...................................................... 26
圖 5、 腸出血性大腸桿菌Verotoxin之VT-I-1、VT-I-2、 VT-II-1、VT-II-2選殖入pGEM®-T easy vector之 限制酶切割確認重組質體DNA電泳圖......................................... 27
圖 6、 腸出血性大腸桿菌Verotoxin之VT-I-1、VT-I-2、
VT-II-1、VT-II-2經限制酶切割後所作之膠體純化
電泳圖............................................................................................... 28
圖 7、 腸出血性大腸桿菌之VT-I-1、VT-I-2、VT-II-1、
VT-II-2選殖pET32a之限制酶切割確認重組質體
DNA電泳圖...................................................................................... 29
圖 8、 腸出血性大腸桿菌VT-I之胺基酸序列比對分析圖....................... 30
圖 9、 VT-I-1（a圖）與VT-I-2（b圖）之胺基酸相似相異
度比對圖........................................................................................... 30
圖10、 腸出血性大腸桿菌VT-II之胺基酸序列比對分析圖..................... 31
圖11、 VT-II-1（a圖）與VT-II-2（b圖）之胺基酸相似 相異度比對圖................................................................................... 31
圖12、 腸出血性大腸桿菌Verotoxin之VT-I-1、VT-I-2、
VT-II-1、VT-II-2表現6小時之粗萃蛋白.......................................... 33
圖13、 以不同時間收集之VT-I-1表現蛋白............................................... 34
圖14、 以不同時間收集之VT-I-2表現蛋白................................................ 35
圖15、 以不同時間收集之VT-II-1表現蛋白............................................. 36
圖16、 以不同時間收集之VT-II-2表現蛋白.............................................. 37
圖17、 腸出血性大腸桿菌Verotoxin之VT-I-1、VT-I-2、
VT-II-1、VT-II-2純化蛋白................................................................ 38
圖18、 腸出血性大腸桿菌Verotoxin之VT-I B及VT-II B
PCR產物電泳圖............................................................................... 40
圖19、 腸出血性大腸桿菌Verotoxin之VT-I B及VT-II
B選殖入pGEM®-T easy vector之限制酶切割確
認重組質體DNA電泳圖................................................................. 41
圖20、 腸出血性大腸桿菌Verotoxin之VT-I B、VT-II B
及pGEX 5X-3以限制酶切割純化結果.......................................... 41
圖21、 腸出血性大腸桿菌Verotoxin之VT-I B、VT-II B
選殖入pGEX 5X-3所作之限制酶切割確認重組質
體DNA電泳圖................................................................................. 42
圖22、 腸出血性大腸桿菌Verotoxin之VT-I B、VT-II B
選殖入pGEX 5X-3誘導表現6小時蛋白之蛋白質
電泳圖............................................................................................... 43
圖23、 以不同時間收集之重組VT B次單位表現蛋白............................. 44
圖24、 重組蛋白VT-I-1、VT-I-2、VT-II-1、VT-II-2之西
方轉漬分析結果............................................................................... 45
圖25、 重組蛋白VT-I B、VT-II B之西方轉漬分析結果........................... 46
圖26、 重組蛋白VT-I-1、VT-I-2、VT-II-1、VT-II-2誘導
之抗體分別中和VT-I及VT-II結果................................................ 48
表 1、 PCR引子對之序列........................................................................... 15

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