臺灣博碩士論文加值系統

實驗室曾自本地的一株Shigella flexneri菌株的基因庫中選殖出一個與大腸桿菌素colicin Ib基因只有一個鹼基對不同的基因，命名為Col Ib基因。雖然一般認為於產生大腸桿菌素的菌中，大腸桿菌素會與一個稱為免疫蛋白(immunity protein)的小蛋白互相作用，使該菌不會受到所產生的大腸桿菌素的毒害；但是實驗室將Col Ib基因選殖到載體後，轉型到不具有免疫蛋白基因的大腸桿菌株中，轉型株卻可利用Col Ib本身的啟動子以mitomycin C誘導，產生具有抑菌活性的Col Ib蛋白，並且誘導前後菌株皆生長良好。

本研究首先將Col Ib基因選殖到不同的選殖載體，轉型到大腸桿菌中，測試轉型株於不同培養及誘導條件的抑菌活性及其中Col Ib蛋白的表現。結果顯示: Col Ib基因選殖於高套數的T-vector質體，並且使表現出的Col Ib蛋白的C端接上his tag (稱為col Ib-c-his蛋白)的轉型株，以300 mL錐形瓶培養100 mL的隔夜菌液，再以mitomycin C誘導六小時，可得到最佳的抑菌活性。進一步將誘導後的菌體，以超音波破菌後，以Ni管柱純化出col Ib-c-his蛋白。取少量蛋白液體冷凍乾燥成粉末後，測試粉末的保存條件及耐熱性。結果顯示: col Ib-c-his蛋白粉末於 4℃保存56天、於60℃下20分鐘、或80℃下5分鐘，完全不失其抑菌活性。

以SDS-PAGE和西方雜配分析所純化的col Ib-c-his蛋白液體，發現其中除了有全長col Ib-c-his蛋白還有一些具有his tag的衍生分子。再將col Ib-c-his蛋白液體通過superdex管柱又可分離出不具有his tag的55 kD和35 kD的蛋白。In vitro實驗證明此二蛋白皆可增加col Ib-c-his蛋白的抑菌活性。55 kD蛋白經質譜鑑定為Alkyl hydroperoxide reductase subunit F；35 kD蛋白經質譜鑑定為Chaperone Hsp 31 glyoxalase 3或Galactitol-1-phosphate dehydrogenase。測試col Ib-c-his基因在這三個蛋白基因突變株的抑菌活性，發現與野生株相比，不論是否誘導，Alkyl hydroperoxide reductase subunit F和Chaperone Hsp 31 glyoxalase 3基因缺失株的上清液活性增加13%，Alkyl hydroperoxide reductase subunit F基因缺失株則只有在未誘導情況下，菌體抑菌活性增加13%。Galactitol-1-phosphate dehydrogenase基因缺失株，不論是否經誘導，上清液與菌體抑菌活性與野生株相似。

以native PAGE分析純化的col Ib-c-his蛋白，發現其中有至少八種不同分子量 (< 20 kD至約260 kD) 的蛋白分子。分別測試這些蛋白分子的抑菌活性，發現只有約260 kD的蛋白分子具有抑菌活性。此約260 kD的蛋白分子經質譜鑑定後，發現其中除了col Ib-c-his外還有6種蛋白，分別為Catalase HPII、P-protein、Galactitol-1-phosphate dehydrogenase、Pyruvate kinase II、DNA gyrase subunit A和
Beta-galactosidase。同樣測試col Ib-c-his蛋白在這六個蛋白基因突變株的抑菌活性，發現與野生株相比，P-protein基因缺失株不論是否經誘導，其破菌液喪失99%的抑菌活性，而其他五個蛋白的缺失株則沒有差異。col Ib-c-his在P-protein基因缺失株不會形成約260 kD的蛋白分子，但是形成一約280 kD的大分子。西方雜配亦顯示col Ib-c-his於野生株與P-protein基因缺失株中的蛋白量幾乎相似。因此本研究可得到一推論:在菌體內col Ib-c-his先與P-protein形成一複合體，再隨機和其他6種蛋白的一或數種緊密結合形成一約260 kD的聚合體，此聚合體會再與 55 kD和35 kD的蛋白以較不緊密的方式結合成更大的分子。此大分子自菌體釋出或進入目標菌時，col Ib-c-his會自聚合體或大分子分開，猜測這就是col Ib-c-his蛋白不會對原生菌產生毒害的原因。

Previously, our laboratory isolated a gene from genomic library of a Taiwan Shigella flexneri strain. The gene has only one nucleotide different from reported colicin Ib gene and was named Col Ib. It has generally been accepted that inside bacteria colicin interacts with a small protein called immunity protein and thus poses no harm to the bacteria. However, our laboratory has cloned the Col Ib gene into a vector and successfully transformed the clone into E. coli strains not carrying the immunity protein gene. The transformants grew well and the Col Ib protein gene could be induced by mitomycin C to produce functional Col Ib protein.
In this study, the Col Ib gene was cloned into different vectors. The E. coli transformants were grown under various conditions and the bactericidal activity and Col Ib expression were examined. The clone with highest activity and Col Ib expression was Col Ib gene cloned into high-copy T vector with his-tag in the C-terminus of the expressed Col Ib protein. The expressed protein is called Col Ib-C-his protein. The growth condition with highest activity and Col Ib expression was transformants grown overnight in 100 mL medium in 300 ml flask and induced by mitomycin C for 6 hours. After induction, the bacterial pellet was sonicated and the Col Ib-C-his protein purified by affinity chromatography with Ni column. The purified protein was freeze-dried and the powder was found to retain full bactericidal activity if stored at 4℃ for 56 days or heated at 60℃for 20 min or 80℃for 5 min.
SDS-PAGE and western analysis revealed that the purified Col Ib-C-his protein solution contained the full-length Col Ib-C-his protein as well as small molecular weight derivatives. Superdex column chromatography of the protein solution further purified 55 kD and 35 kD proteins. The former was identified by mass spectrometry to be Alkyl hydroperoxide reductase subunit F and the later Chaperone Hsp 31 glyoxalase 3 or Galactitol-1-phosphate dehydrogenase. In vitro experiment demonstrated that both 55 kD and 35 kD proteins enhanced the activity of the Col Ib-C-his protein. However, in vivo experiment using knockout mutants of the three identified genes obtained seemingly opposite results.
Native PAGE analysis revealed that the purified Col Ib-C-his protein solution contained at least eight protein complexes of different molecular weight, from < 20 kD to 260 kD. Only the complex of 260 kD has the bactericidal activity. Mass spectrometry identified six proteins in addition to Col Ib-c-his protein. They were catalase HPII, P-protein, galactitol-1-phosphate dehydrogenase, pyruvate kinase II, DNA gyrase subunit A and beta-galactosidase. In vivo experiments using the knockout mutants of these six genes revealed that P-protein was essential for the bactericidal activity of Col Ib-c-his and the other five had no effects. Thus, it is concluded that in E. coli, Col Ib-c-his first formed a complex with P-protein, then formed a 260 kD complex with one or several of the other five proteins. This 260 kD complex may then loosely bind the 55 kD and 35 kD proteins to form a bigger complex. Within bacteria, the 260 kD complex and the bigger complex would not pose any toxicity to the bacteria. Once the 260 kD complex or the bigger complex is released from bacteria, only Col Ib-c-his is taken up by the target bacteria and express the killing activity.

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、 轉型作用 9
五、 DNA 限制酶反應 9
六、 質體 DNA 的 PCR 反應 9
七、 DNA 純化回收 10
八、 重組質體構築 10
九、 以 IPTG 或 mitomycin C 蛋白表現 11
十、 製備供 SDS-PAGE 菌體蛋白、液態蛋白、粉末蛋白 11
十一、 定量抑菌活性 11
十二、 收取培養 2 L (10 L) 隔夜菌液，以 mitomycin 誘導六小時的菌體 pellet……………………………………………………... 13
十三、 利用 ÄKTA prime 純化 col Ib-c-his 蛋白 13
十四、 利用蠕動幫浦純化 col Ib-c-his 蛋白 14
十五、 15% (10%) SDS-PAGE 14
十六、 Coomassie blue 染色 15
十七、 Sliver stain 15
十八、 Western blot 16
十九、 Western hybridization 16
二十、 蛋白透析 17
二十一、 蛋白定量 17
二十二、 製備蛋白粉末 18
二十三、 蛋白溶液或蛋白粉末抑菌活性測定 18
二十四、 8% native gel 19
二十五、 質譜分析 19
結果 20
I. 利用實驗室已有的pQE70-colIb-c-his-ok/BW25113 (附圖八)菌株，純化並定性col Ib-c-his 蛋白 (稱為QE蛋白) 20
(1) 純化 col Ib-c-his 蛋白 20
(2) col Ib-c-his蛋白量與抑菌活性的檢量線 21
(3) col Ib-c-his蛋白粉末較蛋白液體適合長期保存 21
(4) col Ib-c-his蛋白粉末可耐高溫60℃，20分鐘或 80℃，5 分鐘 21
II. 找出經誘導後有最高抑菌活性的菌株 (40 ml菌液培養於300 ml錐形瓶)….. 22
(1) 比較IPTG誘導和以mitomycin C的誘導結果 22
(2) 比較col Ib-C-his和col Ib-N-his的抑菌活性 22
(3) 收集及構築可產出col Ib-C-his的菌株 23
(4) 比較攜帶有高套數的col Ib-C-his質體和低套數的col Ib-C-his質體不同菌株的抑菌活性 23
(5) 比較pT-ColIb-c-his-ok/BW25113不同條件下的抑菌活性 24
(6) 比較pT-ColIb-c-his-ok/BW25113以不同容積培養不同體積的菌液抑菌活性… 24
(7) pT-ColIb-c-his-ok/BW25113以5 L發酵槽培養菌液的抑菌活性 24
III. 利用pT-colIb-c-his-ok/BW25113菌株，純化並定性 col Ib-c-his 蛋白 (稱為 T蛋白) 25
(1) 純化 col Ib-c-his 蛋白 25
(2) SDS-PAGE分析純化的col Ib-c-his蛋白(T蛋白) 25
(3) Native gel分析純化的col Ib-c-his蛋白 (T蛋白和QE蛋白) 27
IV. 利用pT-colIb-c-his-ok/BW25113 (pheA::km)菌株，純化並定性 col Ib-c-his蛋白 (稱為P蛋白) 28
(1) 純化 col Ib-c-his 蛋白 28
(2) pheA 突變株中，col Ib-c-his蛋白中280 kD蛋白，不具有抑菌活
性.............................................................................................................. 29
討論 30
參考文獻 34
表 38
圖 49
附表 77
附圖 79
附錄 89

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