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研究生:呂筱萱
研究生(外文):Xiao-Xuan Lu
論文名稱:大腸桿菌素 colicin Ib 在大腸桿菌中之表現、分布及其活性分析
論文名稱(外文):Expression, localization and functional assay of colicin Ib in Escherichia coli
指導教授:陳建華陳建華引用關係
指導教授(外文):Jiann-Hwa Chen
口試委員:許萬枝胡小婷王雯靜
口試委員(外文):Wan-Jr SyuShiau-Ting HuWen-Ching Wang
口試日期:2015-07-31
學位類別:碩士
校院名稱:國立中興大學
系所名稱:分子生物學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:中文
論文頁數:105
中文關鍵詞:大腸桿菌素
外文關鍵詞:Colicin Ib
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大腸桿菌素 (colicin) 是由一些大腸桿菌株所生產的細菌素,會殺死其他大腸桿菌株。大多數的大腸桿菌素是由大腸桿菌內的質體所負責產生,該質體上還會帶有與大腸桿菌素相對應的免疫蛋白 (immunity protein) 基因。我們實驗室曾自 Shigella Flexneri 的 fosmid library 中篩選到一具有大腸桿菌素 colicin Ib 基因 (cib) 的clone。

本研究首先構築負責表現Col Ib (colicin Ib)、ColIb-C-his (colicin Ib的C端接上 his tag) 及 ColIb-N-his (colicin Ib的N端接上his tag) 的三個 cib 質體。此三質體可成功轉型到不具其免疫蛋白的 DH5α (recA-)、BW25113 (recA+) 及 tonB、exbB 或exbD 基因缺失的 BW25113 中。比較 cib 質體這些轉型株的抑菌活性,發現 tonB、exbB 與 exbD 三基因不會影響菌中 Col Ib 的抑菌活性,但是會減弱 Col Ib 分泌到 cell-free 培養液中。SDS-PAGE 證明三個 cib 質體的 BW25113 轉型株可經 mitomycin C 誘導大量表現全長的 ColIb、ColIb-C-his 及 ColIb-N-his 蛋白;但是西方雜配卻發現,表現 ColIb-C-his 的轉型株經誘導後,除了大量表現全長的蛋白外,還有許多分子量較小的 ColIb-C-his 蛋白也會產生。

菌蛋白 fractionation 及西方雜配實驗顯示:負責表現 ColIb-C-his 的 cib 質體的 BW25113 轉型株,未誘導、經誘導、與隔夜菌液,細胞質皆存在有全長與小分子的 ColIb-C-his 蛋白。另外,未誘導的菌液內膜還有小分子的 ColIb-C-his。經誘導的菌液內膜與外膜有小分子的 ColIb-C-his;間質有全長 ColIb-C-his 蛋白。而經誘導與隔夜的菌液 cell-free 培養液中皆有全長的 ColIb-C-his 蛋白。此結果顯示:轉型株未誘導時,全長的 ColIb-C-his 蛋白主要在細胞質,誘導後可以穿過內膜到間質,甚至再穿過外膜到菌體外。但是小分子的 ColIb-C-his 無法穿過內膜或外膜,而停留在內外膜上。而相同菌的隔夜菌液,只有細胞質中有全長及小分子的 ColIb-C-his 蛋白,但是細胞質、內膜與外膜 fraction 皆有很高的抑菌活性,顯示內膜或外膜上應還有另一種不具 his-tag 的小分子 ColIb-C-his 蛋白。

構築負責表現 ColIb-C-β-lactamase (colicin Ib的 C 端接上bla) 及 ColIb-N-β-lactamase (colicin Ib的 N 端附近接上 bla) 的兩個 cib 質體,並且成功轉型到不具其免疫蛋白的 DH5α 中。發現只有負責表現 colIb-C-β-lactamase 的 cib 質體轉型株可於含有 ampicillin 的培養基生長,另一質體轉型株不能。以 egfp 代替 bla 進行同樣的構築,比較負責表現 ColIb-N-EGFP 的 cib 質體轉型株與負責表現 ColIb-C-EGFP 的 cib 質體轉型株,二者菌落的螢光,發現前者較後者高。此結果顯示 cib 質體轉型株內膜上的小分子 Col Ib (或 ColIb-C-his、ColIb-N-his) 蛋白為一穿膜蛋白,N 端在細胞質,C 端在間質。


Colicin is a toxin protein produced and secreted by some strains of E. coli, and inhibits the growth of other E. coli strains. Most colicins are encoded by plasmid which also carries the gene for its corresponding immunity protein. Previously in our laboratory, a fosmid clone of Shigella flexneri was identified to contain the colicin Ib gene, cib, and its immunity protein gene. In this study, three cib plasmids for expression of ColIb (colicin Ib), ColIb-C-his (colicin Ib with his tag at C terminus), and ColIb-N-his (colicin Ib with his tag at N terminus) were constructed and successfully transformed into DH5α (recA-), BW25113 (recA+), and the tonB-, exbB- and exbD- mutants of BW25113, despite no corresponding immunity protein gene in the transformants. By assaying the inhibition activities of the culture supernatants and the bacterial cell pellets of these transformants, it was found tonB, exbB and exbD genes were required for secretion of colicin Ib, but not for its inhibition activity. SDS-PAGE demonstrated that expression of intact Col Ib, ColIb-C-his and ColIb-N-his could be induced by mitomycin C with cultures of the three BW25113 transformants. Western analysis, however, indicated that truncated forms of ColIb-C-his were also induced in the culture of the transformant carrying the cib plasmid for ColIb-C-his.

Bacterial cell fractionation and western analysis were carried out with non-induced, induced and overnight cultures of BW35113 transformants carrying the cib plasmid for ColIb-C-his. The cytoplasmic fractions of all of the three cultures contained the intact and truncated forms of ColIb-C-his proteins. In addition, the inner membrane fraction of the non-induced culture, and the inner and outer membrane fractions of the induced cultures contained the truncated forms, and the periplasmic fraction of the induced culture contained the intact form. The culture supernatants of the induced and the overnight cultures, but not the non-induced culture, contained the intact form of ColIb-C-his. It appeared that the cytoplasmic intact form could pass the inner membrane to the periplasm, and further pass the outer membrane to the culture medium, while the cytoplasmic truncated forms could only enter and stay in inner or outer membrane. Although no his-tag signals were detected in the inner and outer membrane fractions of the overnight culture, both fractions however contain high inhibition activities, indicating that other truncated forms of ColIb-C-his that lost the C-terminus were present in the membranous fractions of the overnight cultures.

Two cib plasmids for expression of ColIb-C-β-lactamase (bla linked to the C terminus of Col Ib ) and ColIb-N-β-lactamase (bla linked to the vicinity of the N terminus of Col Ib) were constructed and transformed into DH5α successfully. Only the transformant of the former plasmid demonstrated growth on the ampicillin-containing plate. Furthermore, two other cib plasmids were constructed similarly except that bla was replaced by egfp. Colonies of the transformant carrying the cib plasmid for ColIb-N-EGFP (egfp linked to the vicinity of the N terminus of Col Ib) demonstrated higher green fluorescence than colonies of the transformant carrying the cib plasmid for ColIb-C-EGFP (egfp linked to the C terminus of Col Ib). Therefore, it is concluded that the truncated form of Col Ib (or ColIb-C-his, ColIb-N-his) in the inner membrane was a transmembrane protein with the C terminus in periplasm and N terminus in cytoplasm.


目次
前言………………….…………………..…………………..……………………….….1
實驗材料…………….…………………..…………………..……………………….….6
實驗方法…………….…………………..…………………..………………….……….8
一、 抽取質體 DNA………………………………..…………….……….….…...8
二、 DNA 黏接反應……………...…………………………….………………....8
三、 勝任細胞的製備……………………………………..……...………….…….8
四、 轉形作用………………………………………...…………………...…..…...8
五、 DNA 限制酶反應……………..……………………...……………………...8
六、 質體 DNA 的 PCR 反應……………………...…………………..……….9
七、 DNA 純化回收…………………………………...………………...……......9
八、 收取菌體蛋白…………………………………………………..…………….9
九、 以 mitomycin C 誘導蛋白表現…………………………...………………...9
十、 10% SDS-PAGE…….......……...….………………………….…..….............9
十一、 Coomassie blue 染色………………………………..……….……….10
十二、 Western blot…………………………….....…….…….……….……….10
十三、 Western hybridization…..…………………………….……...………....10
十四、 定量抑菌活性…………………………………..…………...…………11
十五、 重組質體構築……………………..……………..……….…………..12
十六、 Bacterial cell fractionation ………………………………....….…….....13
十七、 菌液上清液的 TCA 沉澱……………………………....….………....14
十八、 ELISA Reader 檢測綠色螢光……………………………....….….......14
十九、 Col Ib 與目標細胞共培養………………………………....….……....14
二十、 以倒立相位差顯微鏡觀察菌落綠色螢光………………………........14

結果………………………………………….....……………………..………………..16
一、 實驗室現有質體之 cib 序列有誤,重新構築序列無誤的 cib 質體….....16
二、 菌液 cell free 上清液及菌體破菌液的抑菌活性的定量方法……....……16
1. 上清液的抑菌活性的定量方法…………...………….………...……..16
2. 菌體破菌液的抑菌活性的定量方法………...……..…….…………16
(a) 破菌方法…………………………………….……..……………..16
(b) 破菌前將菌體回溶的溶液……………….……..………………..17
三、 比較 pQE70-ColIb-C-his-ok/DH5α 及 pQE70-ColIb-C-his/DH5α 的抑菌活性 (二者只有 cib 兩個鹼基的差異)……………………………...….17
四、 具有 cib 質體的菌 cib 基因表現受到 mitomycin C 的誘導而增加…..17
1. 預測三個 cib 質體 cib promoter 附近的 SOS box…………….…17
2. 具有 cib 質體的菌可經 mitomycin C 誘導而大量表現全長及分子量較小的 Col Ib 蛋白……….…………………….………………….…18
3. Col Ib 蛋白表現會使菌的生長減緩…………….……….……….…..18
4. ColIb-C-his 蛋白量會隨 mitomycin C 誘導時間增加而增加……18
五、 Col Ib 的 N 端有 his tag 會降低 Col Ib 的抑菌活性,但是 C 端有 his tag 則不影響其抑菌活性……………….…..………..………..…………19
1. 三個具有 cib 質體的菌經 mitomycin C 誘導後上清液及破菌液抑菌活性會增加………………………………….…………….……………19
2. 經 mitomycin C 誘導後,表現 ColIb-C-his 的 cib 質體轉型株,其菌蛋白表現量較表現 Col Ib 的 cib 質體轉型株的菌蛋白表現量多;而後者菌蛋白的表現量又較表現 ColIb-N-his 的 cib 質體轉型株的菌蛋白表現量多…………………………………………………..……19
六、 phoA、endA1、fhuA2、recA1、relA1、thi-1 不會影響具有 cib 質體的菌,其上清液及破菌液的液菌活性……..………...……………………………19
1. 表現不具 his tag 的 Col Ib 的 cib 質體 DH5α 轉型株的上清液抑菌活性,比相同質體的 BW25113 轉型株的上清液抑菌活性高…...19
2. phoA、endA1、fhuA2、recA1、relA1、thi-1 不會影響具有 cib 質體的菌,其上清液及破菌液的液菌活性………………………………..20
七、 tolC、tufA、tufB 不會影響帶有cib 質體的菌的抑菌活性……..…..………20
八、 tonB、exbB 與 exbD 會影響具有 cib 質體的菌分泌 Col Ib 或 ColIb-C-his,但 cirA 不會影響………………………….……...………….20
九、 ColIb-C-his 蛋白於生產菌株中存在的位置.………………..….…………20
1. 經誘導後所得到的全長 ColIb-C-his 蛋白主要在間質與細胞質中,經誘導後所得到的 ColIb-C-his 衍生分子主要在細胞質、內膜與外膜中,但是隔夜菌液中,全長 Col Ib 蛋白及 ColIb-C-his 衍生分子主只存在細胞質中….……..…………..………………..……….....…..…..…20
2. 收取的間質、細胞質、內膜和外膜四個 fraction 之檢驗……….......21
3. 全長的 ColIb-C-his 及 ColIb-C-his 衍生分子可能都具有抑菌活性…………..………………..………………..……………………..…..21
4. 隔夜及經誘導的菌主要會分泌全長的 ColIb-C-his 至胞外,經誘導的菌也會分泌小量 ColIb-C-his 衍生分子,而未經誘導的菌則沒有任何形式的 ColIb-C-his 至胞外………..…………………....…..………21
十、 Col Ib 於生產菌株膜上的方向……………….………………………..…..22
1. 隔夜培養的生產菌株菌落,其Col Ib 的 C 端會位於間質中,N 端會位於細胞質中………………………………………………….…..22
2. 經 mitomycin C 誘導後,生產 ColIb-C-EGFP 的菌,其破菌液綠色螢光會較生產ColIb-N-EGFP 的菌之破菌液強……………….……..23
3. 經 mitomycin C 誘導後,生產 Col Ib 的 C 端具有 EGFP 的菌,其細胞質與內膜 fraction 表現綠色螢光………………………...………………..23
4. 所收取的含 ColIb-C-his 之 細胞質 fraction 與 BW25113 共培養後,會造成 BW25113 無法生長…....…………………….……………………..24

討論.…………………..…………………..……………..………...……….…………..25
參考文獻.…………………..…………………..…………………..…………….….…30
表.…………………..…………………..……………..…………...…….………..……36
圖.…………………..…………………..……………..…………...…….……………..50
附表.…………………..…………………..……………..………...……….…….…….83
附圖.…………………..…………………..……………..………...……….…………..84
附錄………………..…………………..……………..……………...….……….……..97


表目次
表一:本研究所使用的菌種……..…………..……..………………….………………36
表二:本研究所使用的質體……….………………..………………......……..………37
表三:研究所使用的引子……..…………………..……………..…...………..………40
表四:找出菌液上清液抑菌活性的定量方法……..…………..…….……….…….…41
表五:菌液經二種不同離心條件後,菌 pellet 的菌數………...……………….…..41
表六:找出菌液菌體破菌液抑菌活性的定量方法 (a)破菌方法..…….…………..…42
表七:找出菌液菌體破菌液抑菌活性的定量方法 (b)破菌前將菌體回溶的溶液….42
表八:比較 pQE70-ColIb-C-his-ok/DH5α 及 pQE70-ColIb-C-his/DH5α 上清液及破菌液的抑菌活性……...………………...………...………...………..…..….…43
表九:三個具有 cib 質體的菌經 mitomycin C 誘導生長減緩………...……..……43
表十:C 端有 his tag 的 cib 質體的菌抑菌性與沒有 his tag 的 cib 質體的菌抑菌活性相同…..…………………..…………………………...………….……….44
表十一:具有 cib 質體的 DH5α,將 Col Ib 分泌出去的能力較具有 cib 質體的 BW25113 強...…...…...………...……….……...……….……...……..….……45
表十二:phoA、endA1、fhuA 、recA、relA1、thi-1 不會影響帶有 cib 質體 的 BW25113 分泌 Col Ib 或 ColIb-C-his……………….…………...…………45
表十三:tolC、tufA、tufB 不會影響帶有 cib 質體 的 BW25113 分泌 ColIb-C-his…………………………………………………..…………………46
表十四:tonB、exbB 與 exbD會影響具有 cib 質體的菌分泌 Col Ib 或 ColIb-C-his,但 Cir 不會影響…......………………………………….…………….………46
表十五:全長的 ColIb-C-his 及 ColIb-C-his 的衍生分子可能都具有抑菌活性…………………………………………………………………….…....……47
表十六:菌中 Col Ib 蛋白帶有 EGFP tag 時,經 mitomycin C 誘導後菌體中可顯示綠色螢光…………….……....…………………………………...………….47
表十七:經 mitomycin C 誘導後,生產 Col Ib 的 C 端具有 EGFP 的pQE70-ColIb-C-EGFP-his-ok/BW25113,其細胞質 fraction 表現綠色螢光………….……….……….……….……...………………………………….48
表十八:所收取的 pQE70-ColIb-C- his-ok/BW25113 細胞質 fraction 及 pQE70-ColIb-C-EGFP-his-ok/BW25113 細胞質 fraction 與BW25113共培養後,菌的綠色螢光讀值…………………………………....………..………….48
表十九:pQE70-ColIb-C-his-ok/DH5α 及 pQE70-ColIb-C-his/DH5α鹼基、胺基酸、抑菌活性差異………………………...……....………....………..………….49



圖目次
圖一:ColIb-T-1 構築圖……………....…..…………...………………………………50
圖二:ColIb-T-2 構築圖……………....………………..……………………………...51
圖三:pQE70-ColIb-C-his-R1構築圖.......………………...…………………………...52
圖四:pQE70-ColIb-ok 構築圖....…………………………...………………………...53
圖五:pQE70-ColIb-N-his-ok 構築圖...….…………………...………..……………...54
圖六:pQE70-ColIb-C-his-ok 構築圖....……..………………...……………………...55
圖七:pQE70-ColIb-C-EGFP-his-ok 構築圖.……..…………………...……………...56
圖八:pQE70-ColIb-N-EGFP-his-ok 構築………….…………………...…………….57
圖九:pQE70-ColIb-C-β-his-km-ok 構築圖…………………………...………..…….58
圖十:預測三個 cib 質體 cib promoter 附近的SOS box……..……...……………59
圖十一:具有 cib 質體的菌可經 mitomycin C 誘導表現 Col Ib 蛋白…….……..60
圖十二:Col Ib 蛋白 C 端帶有 his tag 的 cib 質體,其 ColIb-his 蛋白量隨mitomycin C 誘導的時間增長而增加……………………………….……….62
圖十三:C 端或 N 端帶有 his tag 的 cib 質體,經 mitomycin C 誘導後,Col Ib 蛋白的表現量之比較.…………………..………………………..……………64
圖十四:經誘導後所得到的全長 Col Ib 蛋白主要在間質與細胞質中,經誘導後所得到的Col Ib 衍生分子主要在細胞質、內膜與外膜中,但是隔夜菌液中,全長 Col Ib 蛋白及 Col Ib 衍生分子只存在細胞質中………...…………..65
圖十五:所收取的細胞質 fraction 有些微的間質 fraction污染…..………..…...….68
圖十六:所收取的內膜和外膜 fraction有些微的細胞質 fraction 污染……..….….70
圖十七:所收取的細胞質和外膜 fraction 有內膜 fraction 污染………….……….72
圖十八:所收取的細胞質 fraction 有些微的外膜 fraction污染…………...……….74
圖十九:只有隔夜及經誘導的菌液中有全長的 ColIb-C-his 分泌至胞外,未經誘導的菌液則沒有……………………………………………...………..…………76
圖二十:生產菌株隔夜菌落中的 Col Ib,其 C 端會位於間質中,N 端會位於細胞質中………………………………………………………………….…………77
圖二十一:經 mitomycin C 誘導後,生產 Col Ib 的 C 端具有 EGFP 的 pQE70-ColIb-C-EGFP-his-ok/BW25113,其細胞質 fraction 呈現明顯綠色…....………………………………………………….....……………………79
圖二十二:BW25113 與 pQE70-ColIb-C-his-ok/BW25113 誘導後菌液的細胞質 fraction 共培養,BW25113 會死亡.……………………….…………….……80
圖二十三:以 ColIb-C-his 生產菌株建立的可能 Col Ib 型態與分佈模型…….………………….………………………….……….…………….……81
圖二十四:隔夜培養生產菌落中,Col Ib 蛋白位於膜上時的可能方向……………82

附表圖目次
附表一:大腸桿菌素的分類……..…………………..………………...………..……83
附圖一:大腸桿菌素進入敏感菌株後易位的方式……....……………..……….……84
附圖二:大腸桿菌素的模塊化結構域……..………..…………..……….……………85
附圖三:腸桿菌素進入敏感菌株的機制……..……...……..……..………..…………86
附圖四:Colicin Ia 與 Colicin Ib胺基酸序列比較結果……..………..………...……87
附圖五:Colicin Ia 結構圖……..……...……..……..…….. ..…..………..…………88
附圖六:pQE70-ColIb-C-his……..………………..…..………………...…..…………89
附圖七:pQE70-ColIb-N-his……..………………..…..……………….....……………90
附圖八:pACYC184-imm………………….………………...………...………………91
附圖九:pColIb-T………………..………………....………...………...………………92
附圖十:pQE70-colIb-C-β-his-km……….………...…..……………….....……………93
附圖十一:pQE70-colIb-N-β-his-km…………..…….......……………….....…………94
附圖十二:DAS 軟體對於 Col Ib 蛋白序列是否具有穿膜性質的分析………95
附圖十三:pQE70………………..…………………...…..……………….....…………96


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