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研究生:王大元
研究生(外文):Ta-Yuan Wang
論文名稱:膿腫分枝桿菌CS1c細胞質外功能σ因子(mab4459c)與σ聯繫蛋白(mab4454c)之特性研究
論文名稱(外文):characterization of extracytoplasmic function sigma factor (mab4459c) and sigma-associated protein (mab4454c) in Mycobacterium abscessus CS1c.
指導教授:胡小婷
指導教授(外文):Shiau-Ting Hu
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:微生物及免疫學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:78
中文關鍵詞:σ因子σ聯繫蛋白膿腫分枝桿菌CS1c
外文關鍵詞:extracytoplasmic function sigma factorsigma-associated proteinMycobacterium abscessus CS1c
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膿腫分枝桿菌(Mycobacterium abscessus)為革藍氏陽性菌,會感染人體許多部位,包括肺部、皮膚、四肢和軟組織等,造成慢性肺部疾病、皮膚病、中耳炎等病徵。先前文獻已觀察到膿腫分枝桿菌臨床菌株390會有兩種不同菌落形態,分別是平滑型(smooth)和粗糙型(rough),這兩種形態經過數代培養後,型態會互相轉換,其轉換率為10-5~10-6,粗糙型較具毒性。本實驗室於先前研究亦發現膿腫分枝桿菌臨床菌株CS1c會形態轉換,首先會從光滑透明型(smooth transparent;SmT)轉變為粗糙型(R1),再轉變成光滑不透光型(smooth opaque;SmO),最後又轉回粗糙型(RR2) 其轉換率約為10-4~10-5。平滑型和粗糙型最主要之差異在於醣類鍵結物(glycopeptidolipid;GPL)含量, GPL是透過GPL基因組調控合成,我們在GPL基因組內找到ecf和sap兩調節基因,並證明兩基因轉譯之Ecf和Sap蛋白會交互作用,Ecf為細胞質外功能σ因子(extracytoplasmic sigma factor),Sap為σ聯繫因子(sigma associated protein)。Ecf會加強轉錄GPL基因組的部份基因,包含自身ecf、sap和pks的啟動子,但對mmpS4啟動子則有負調控之現象,同時加入Ecf和Sap蛋白後,基因轉錄活性有下降趨勢,sap可能抑制受Ecf調控之轉錄作用。我們大量表現Ecf蛋白,使其正調控GPL產量,但無法使膿腫分枝桿菌粗糙型R1轉變成平滑型菌落,Ecf和Sap蛋白會調控GPL部分基因但無法改變形態。細胞質外功能σ因子會在不同生長點和適當壓力下活化表現,我們在恥垢分枝桿菌(Mycobacterium smegmatis)實驗可知ecf啟動子會在指數期(log phase)有較高轉錄結果,但目前尚無法找到適當之壓力刺激,亦無法構築可在膿腫分枝桿菌實驗之適當報導載體(reporter vector),對於Ecf加強轉錄其它啟動子、Ecf受何種壓力刺激與菌落形態轉換之機制仍需後續之研究。
Mycobacterium abscessus is an increasingly important Gram-positive bacterium that can cause a wide spectrum of human infections, such as lung, skin, soft tissue and limbs infections. In earlier study by Thomas, it had been observed that Mycobacterium abscessus clinical strain 390 can be isolated as smooth and rough colony. Both of morphologies can reverse during serial passages. The spontaneously morphologic reversion rate was about 10-5~10-6 and the rough colony was more virulent than the smooth colony. In earlier study by our laboratory, we also analyzed the colony morphology variation of clinical strain CS1c. The CS1c colony morphology can switch from smooth transparent (SmT) to rough (R1), from rough (R1) to smooth opaque (SmO), and from smooth opaque (SmO) to rough (RR2) again. The CS1c morphologic reversion rate was about 10-4~10-5. The major difference between smooth and rough morphology was glycopeptidolipid (GPL) content. GPL was synthesized and regulated by GPL locus. It was predicted that two genes in GPL locus, ecf (mab_4459c) and sap (mab_4454c) may regulate GPL synthesis. The ecf that is thought as extracytoplasmic function sigma factor, may express in optimal stress conditions and growth phase, then transcribed other genes. The sap that is thought as sigma-associated protein may participate in initiation step of transcription. In this study, we demonstrated that Ecf can interact with Sap and that exogenous Ecf can enhance the transcription of ecf, sap and pks promoter but inhibit the transcription of mmpS4 promoter. In the presence of Sap, the transcriptional function of Ecf may be inhibited. In order to analyze the mechanism of colony morphology variation, we over-express exogenous Ecf in Mycobacterium abscessus R1, and found the rough colony could not switch to smooth one. Ecf and Sap protein can regulate parts of GPL locus without being involved in morphological switch. It was known that extracytoplasmic function sigma factor can be up-regulated in optimal growth condition and stress stimulation. In this study transcription from the ecf promoter can be enhanced in log phase in Mycobacterium smegmatis, whereas the optimal stress conditions remained investigated.
中文摘要 1
Abstract 2
第一章 緒論 4
1-1快速生長分枝桿菌(Rapidly Growing Mycobacterium;RGM) 5
1-2膿腫分枝桿菌(Mycobacterium abscessus) 6
1-3菌落形態變異(colony morphological variation) 7
1-3-1鳥型分枝桿菌(Mycobacterium avium)菌落形態變異和特性 7
1-3-2恥垢分枝桿菌(Mycobacterium smegmatis)菌落形態變異和特性 8
1-3-3膿腫分枝桿菌(Mycobacterium abscessus)菌落形態變異和特性 9
1-4醣類胺基酸脂質鍵結物(glycopeptidolipid;GPL) 10
1-5 σ70因子(sigma factor)概論 12
1-6細胞質外功能σ因子(extracytoplasmic function sigma factor;Ecf) 13
1-7本文動機及方向(rationales and aims) 14
第二章 材料與方法 16
2-1 藥品、試劑及酵素 16
2-2細菌菌株和儲存方式 (bacterial strain and stock) 16
2-3質體及引子(plasmid and primers) 16
2-3-1 pGB-hES’ 16
2-3-2 pGB-hES 17
2-3-3 pMNOA-lacZpromoterless 17
2-3-4 pMNOA-lacZ-promoter 17
2-3-5 pMNOApra-lacZ-promoter 17
2-3-6 pYUB412a-02A04和 pYUB412a-22A08 18
2-4細菌之培養 18
2-5質體之構築 18
2-5-1聚合酶連鎖反應(Polymerase Chain Reaction, PCR) 18
2-5-2瓊脂凝膠製作及電泳 (agarose gel electrophoresis) 19
2-5-3 限制酶切割(restriction enzyme digestion) 19
2-5-4 DNA之回收及純化 (DNA recovery and purification) 19
2-5-5 DNA接合反應 (DNA ligation) 19
2-5-6大腸桿菌熱休克轉型勝任細胞(heat shock transformation competent cell)之製備 20
2-5-7大腸桿菌之電穿孔勝任細胞製備 (E. coli electroporation competent cell) 20
2-5-8分枝桿菌之電穿孔勝任細胞製備 (Mycobacteria electroporation competent cell) 20
2-5-9細菌轉型(transformation) 21
1.大腸桿菌熱休克法(heat-shock transformation) 21
2.電穿孔法(electroporation transformation) 21
2-6質體之純化 22
2-6-1以Alkaline lysis method少量純化質體DNA ( Miniprepation of plasmid DNA ) 22
2-6-2以套組(Kit)純化少量質體 23
2-7細菌雙雜合試驗 (bacterial two-hybrid assay) 23
2-8乙型半乳糖分解酶(β-galactosidase)活性之測定 24
2-9西方墨點分析 (Western blotting assay) 25
2-9-1 SDS聚丙烯醯氯凝膠製備與電泳(SDS-PAGE preparation and electrophoresis) 25
2-9-2蛋白質轉漬 (transfer) 25
2-9-3免疫抗體標定 26
2-9-4化學冷光呈色 26
2-10膿腫分枝桿菌基因組DNA製備 (preparation of genomic DNA of M. abscessus) 26
2-11大腸桿菌和恥垢分枝桿菌蛋白質表現 27
第三章 結果 28
3-1 ecf、sap具啟動子活性 28
3-1-1 報導基因載體構築(reporter vector construction) 28
3-1-2偵測ecf和sap啟動子活性 28
3-2利用細菌雙雜合系統,證明Ecf與Sap之交互作用 29
3-3質體構築與蛋白表現(plasmid construction) 30
3-3-1受溫度調控(hsp60-promoter)之基因載體構築 30
3-3-2受溫度調控(hsp60-promoter)基因載體之Ecf與Sap之蛋白表現 30
3-3-3構築其它GPL報導基因載體(reporter vector construction) 31
3-3-4偵測其它GPL啟動子活性,分為強啟動子與弱啟動子 31
3-4偵測Ecf和Sap是否可轉錄GPL基因組 31
3-5膿腫分枝桿菌是否受ecf與sap調控導致型態改變 33
3-6細胞質外功能σ因子需受到適當壓力刺激和生長狀態而表現 34
3-6-1 ecf啟動子在恥垢分枝桿菌內之生長曲線與轉錄分析 34
3-6-2 細胞質外功能σ因子在恥構分枝桿菌內會受到何種壓力刺激而表現 35
3-6-3 36
(1) 重新構築適用於膿腫分枝桿菌之報導載體 36
(2)偵測Ecf和Sap在膿腫分枝桿菌是否可轉錄GPL基因組 36
(3)細胞質外功能σ因子在膿腫分枝桿菌內會受到何種壓力刺激而表現 36
第四章 討論 38
第五章 參考文獻 44
第六章 圖表 49
表一、本文所使用之細菌菌株 49
表二、本文所使用之質體 50
表三、本文使用之引子 55
表四、本文所使用之抗體 58
圖一、報導載體之構築 59
圖二、膿腫分枝桿菌GPL locus相對位置 60
圖三、在恥垢分枝桿菌(黑)和大腸桿菌(灰)之GPL啟動子活性 61
圖四、Ecf與Sap蛋白之交互作用 62
圖五、hsp60調控之Ecf和Sap蛋白表現質體與西方墨點分析結果 63
圖六、單獨Ecf或與Sap蛋白共同存在對GPL啟動子轉錄之影響 64
圖七、Ecf蛋白單獨存在對GPL啟動子轉錄之影響 66
圖八、37℃時, ecf啟動子於細菌不同生長期之活性分析 67
圖九、在恥垢分枝桿菌內不同壓力對ecf啟動子表現之影響 68
圖十、構築適用於膿腫分枝桿菌之報導載體 69
附錄 70
附錄一、GPL在膿腫、恥垢和海龜分枝桿菌之相對位置[15] 70
附錄二、恥垢分枝桿菌之GPL合成示意圖[15] 71
附錄三、恥垢、膿腫和海龜分枝桿菌GPL之相似性與功能[15] 72
附錄四、恥垢分枝桿菌與膿腫分枝桿菌細胞質外功能σ因子 (Ecf)之胺基酸序列比對 73
附錄五、恥垢分枝桿菌與膿腫分枝桿菌σ聯繫蛋白(Sap)之胺基酸序列比對 74
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