臺灣博碩士論文加值系統

Part I
以生物質譜法鑑定結核桿菌及觀察其抗藥性

肺結核桿菌(Mycobacterium tuberculosis，Mtb ) 是一株歷史久遠的細菌，當感染這株細菌時，會造成結核病，一般而言，因為分枝桿菌生長緩慢，治療需要蠻長一段時間，但很多人會不按時吃藥甚至是自行停藥，很容易造成細菌突變而產生抗藥性菌株出現，然而在臨床的檢驗藥物抗敏性，須耗費將近2~3個月的時間才可確定感染結果。
本研究希望能透過液相層析質譜技術搭配”亂槍打鳥”(shotgun proteomics)的方法來鑑定Mtb並觀察抗藥性蛋白質表現，希望能縮短一般在臨床上所需要的鑑定時間，也因為Mtb對於一般實驗室，屬於較難取得的細菌，因其會經由飛沫傳染，必須在生物安全等級第三級 ( P3 ) 的實驗室操作，若需從P3實驗室拿出，也一定要滅菌才能拿出來，因此有樣品來源的實驗室不多，在現今的研究領域上這部分還有許多層面可以探討。
shotgun proteomics屬於不需跑膠的偵測蛋白質技術，將全細胞打破後得到蛋白質混合物，再利用酵素將蛋白質混合物消化為胜肽混合物藉由層析管柱分離後進入質譜偵測，由質譜得到的數據再利用生物資訊來分析，比對到的蛋白質序列片段利用blast找出Mtb的特異性片段，利用這樣的片段來確認臨床檢體為Mtb或是NTM。另外建立與抗藥性蛋白質相關的資料庫，觀察抗藥性蛋白的表現。

Part II
以 Bacillus licheniformis 生物合成銀奈米粒子

奈米科技在近年來是很熱門的技術之一，而奈米顆粒也被應用於不同的領域，無論是科學、工業或是醫學…等，都有其應用價值。合成奈米顆粒的方式有很多，其中利用微生物生成奈米粒子是近幾年來發展的重點，雖然在技術層面還有一些問題需要解決，像是生成出來的奈米顆粒品質不一或是無法達到量產的效益…等等。
台東大學廖尉岑老師於台東知本溫泉分出一株新種(Strain)的地衣芽孢桿菌(Bacillus licheniformis)，命名為NTTU-CP4，利用SEM、TEM以及ICP/MS，確認NTTU-CP4同樣具有將銀離子還原成奈米銀的能力，進一步利用LC/MS觀察其蛋白質表現，希望可以觀察到跟氧化還原相關的蛋白質，另外當樣品分別培養於23℃和55℃並且與不同濃度的硝酸銀共培養後，但每個樣品之間的蛋白質表現，並無觀察到有意義的差異。

Part I
Diagnosis of Mycobacterium tuberculosis & drug resistance observation

The Mycobacterium tuberculosis is one of the difficult treatment diseases in the global world. There are many people infected every year especially those immunodeficiency patients. The inadequate infection control would induce the drug resistance Mtb strain. The Mtb that resist to two kinds of most useful first-line medicine (Isoniazid and Rifampicin) always called multi-drug resistance Mtb strain (MDRTB).
Mtb grow very slowly than other bacteria. Therefore, in this research, we wanted to find an easily method which would actually identify the Mtb and decrease the inspection time than traditional tests. Otherwise, we hope that using shotgun proteomics approach would help us to find some kind of proteins that related to the MDRTB. This would possible provid some information to doctors in clinical treatment.
We broke the Mtb cells using mini-beadbeater cell disrupter and cleaned up extracted proteins by filtration and ultra-centrifuge; followed by trypsin digested into peptides. We used homemade tip C18-column connected with tandem MS to separate and detect under data-dependent mode. Using BioworkTM search engine searched against NCBI BLAST database to identify unknown proteins. The results shows there were some specific peptides had been detected. That could help us to identified Mtb or NTM, but to identify MDRTB still not yet.

Part II
Using Bacillus licheniformis Biosynthesized Silver Nanoparticle

Nanotechnology is very popular for its various applications nowadays. For example, the silver nanoparticles (nanosilver) formed by biosynthesis that could be used in medicine as antibiotic, anti-virus activity, anti-inflammatory.
The previous researches reported that AgNO3 solution could be used for synthesis silver nanoparticles, because there are some bacteria that could reduce Ag+ to Ag by nitrate reductase, but the reduction mechanism still didn’t demonstrate accurate yet. A Bacillus licheniformis strain (NTTU-BL1) newly isolated from Taitung hot spring shows a high degree of reduction activity.
In this research, NTTU-BL1 cells were treated with different concentration AgNO3 solutions (10-4, 10-3, 10-2 mM AgNO3) at 23℃ and 55℃, and the nanosilver biosynthesis process was examined by SEM, TEM and ICP/MS. Furthermore, the shotgun proteomic approach was used to identify bacterial proteins related to nanosilver biosynthesis process and silver toxin resistant mechanism.
Using BioworkTM search engine searched against NCBI BLAST database to identify unknown proteins. There are several proteins with function be predicted. The results were toward to two directions, one was to find out what kinds of proteins would participate in the reduction. On the other hand, we tried to find the same/different proteins expression in different samples.

目錄
致謝 II
摘要 III
以生物質譜法鑑定結核桿菌及觀察其抗藥性 III
以 BACILLUS LICHENIFORMIS 生物合成銀奈米粒子 IV
ABSTRACT V
DIAGNOSIS OF MYCOBACTERIUM TUBERCULOSIS & DRUG RESISTANCE OBSERVATION V
USING BACILLUS LICHENIFORMIS BIOSYNTHESIZED SILVER NANOPARTICLE VI
目錄 I
附圖目錄 II
附表目錄 III
ＰＡＲＴ　Ｉ
Diagnosis of Mycobacterium tuberculosis & drug resistance observation
第一章 緒論 1
一、研究肺結核桿菌之重要性 1
1.結核分枝桿菌 1
2.抗藥性結核菌 2
3.臨床檢驗 2
二.蛋白質體學 3
1.蛋白質體學簡介 3
2.質譜儀的應用 4
三.文獻回顧 6
四.研究動機與目的 7
第二章 研究材料與步驟 9
一.實驗藥品與試劑 9
二.儀器裝置 9
三.菌株的收集 10
四.蛋白質萃取 11
五.微波消化 12
六.液相層析-奈米級電噴灑質譜儀 13
七.資料庫的建立 14
八.生物資訊的運用 15
第三章 實驗結果與討論 16
第四章 結論 21
第五章 圖表附錄 22
第六章 參考資料 41
ＰＡＲＴ　ＩＩ
Using Bacillus licheniformis Biosynthesized Silver Nanoparticle
第一章 緒論 47
奈米科技 47
1. 奈米技術簡介 47
2.奈米顆粒 48
二.電子顯微鏡 48
1.電子顯微鏡簡介 48
2.掃描式電子顯微鏡(Scanning Electron Microscope ，SEM) 49
3.穿透式電子顯微鏡（Transmission Electron Microscope，TEM） 49
三.研究動機與目的 50
第二章 研究材料與步驟 51
一.實驗藥品與試劑 51
二.儀器裝置 52
三、菌株來源 53
四、穿透式電子顯微鏡技術(TEM) 54
五、掃描式電子顯微鏡技術(SEM) 55
六.蛋白質萃取 55
七.微波消化 56
八.液相層析-奈米級電噴灑質譜儀 56
九.資料庫的建立 57
十.生物資訊的運用 57
第三章 實驗結果與討論 58
第四章 結論 62
第五章 圖表附錄 63
第六章 參考資料 98
附錄A 101
附錄B 123


附圖目錄
圖 一 電噴灑現象示意圖 5
圖 二 利用shotgun proteomics快速分析結核桿菌流程 8
圖 三、Mycobacterium tuberculosis 獨有的胜肽片段 31
圖 四、Mycobacterium tuberculosis特異性片段二次質譜圖 32
圖 五、Catalase-peroxidase的蛋白質序列 38
圖 六、DNA-directed RNA polymerase subunit beta的蛋白質序列 40
圖 七、掃描式電子顯微鏡(SEM)以及元素分析(EDS) 63
圖 八、掃描式電子顯微鏡(SEM)以及元素分析(EDS)的方法觀察加入硝酸銀共培養的NTTU-CP4。 64
圖 九、穿透式電子顯微鏡(TEM)觀察NTTU-CP4是否有銀元素的存在。 66
圖 一〇、利用無機質譜儀(ICP/MS)來偵測樣品是否有銀的存在。 67
圖 一一、(A)23℃培養Bacillus licheniformis NTTU-CP4搜尋資料庫的結果利用PSORTb v3.0.2預測其蛋白質位置. (B) 55℃培養Bacillus licheniformis NTTU-CP4利用PSORTb v3.0.2預測其蛋白質位置. 95


附表目錄
表 一、 實驗使用菌株其標號、菌名以及來源 10
表 二、液相質譜儀及Data dependent參數設定 13
表 三、nr 資料庫搜尋後找到Mycobacterium smegmatis獨有的胜肽片段之結果 22
表 四、利用TB complex database 搜尋的結果 24
表 五、針對MH+=803.72二次質譜掃描，並將結果搜尋nr資料庫所得之蛋白質資訊 33
表 六、利用Mtb Drug Resistance Database做為資料庫搜尋後得到與抗藥性相關的蛋白質序列 35
表 七、實驗菌株列表 53
表 八、將Bacillus licheniformis NTTU-CP4利用Unipot下載之資料庫做搜尋，所得到的蛋白質結果 68
表 九、將偵測到的所有蛋白質分類 96
表 一〇、將蛋白質功能利用KEGG以及Gene Ontology分類 97

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