臺灣博碩士論文加值系統

結核病是一種目前仍普遍存在於全世界，由結核分枝桿菌(Mycobacterium tuberculosis, MTB)感染所造成的傳染病，主要以影響肺部為主。預防結核病的方法為注射卡介苗(Bacillus Calmette-Guerin, BCG），卡介苗是由減毒活牛結核分枝桿菌（Mycobacterium bovis）製備而成。甘油二酯激酶(diacylglycerol kinases, DAGKs/DGKs)為卡介苗細胞內脂質激酶，在脂質的代謝上，為一極其重要酵素，其功能為利用ATP磷酸化甘油二酯(sn-1,2-diacylglycerol, DAG)，以合成磷脂酸(phosphatidic acid, PA)。甘油二酯與磷脂酸，在生物體內的訊息傳遞上，均扮演著極為重要的角色。DAGKs在原核及真核生物體內都有發現，但是在原核與真核中，此激酶彼此之相似度並不高。目前尚無臺灣使用之BCG Tokyo 172 strain 的DAGK蛋白結構研究。本實驗嘗試探討卡介苗內DAGK之結構，選擇DAGK基因將其基因重組後，送入大腸桿菌菌種以誘導其表現。以SDS-PAGE分析與圓二色光譜(circula dichroism, CD)確認其蛋白質二級結構，得知其含有77.6% α-helix，6.63% β-sheet，6.43% turn與10.66% Random coil。此外，透過其胺基酸序列，經由軟體模擬出其立體構型，並藉由此二組數據比對此甘油二酯激酶分子結構。透過蛋白質結構分析，吾人得以設計新型蛋白質或藉此作為改造已知蛋白質提供可靠依據，抑或是對新型藥物設計提供合理標靶分子構型。

Tuberculosis still remains a global pandemic, due to Mycobacterium tuberculosis, MTB, infection mainly targeting the lungs. One of the important methods for the prevention of tuberculosis, is by vaccination of BCG (Bacillus Calmette-Guerin, BCG), a toxically attenuated live vaccine prepared from bovine tuberculosis (Mycobacterium bovis). Diacylglycerol kinase (diacylglycerol kinases, DAGKs/DGKs), isolated from MTB, is an important enzyme responsible for intracellular lipid phophorylation during the metabolism of lipids. It phosphorylates diacylglycerol (sn-1,2-diacylglycerol, DAG) using ATP to synthesize phosphatidic acid (PA). Diacylglycerol and phosphatidic acid play an important role within organisms. DAGKs have been found to distribute in both prokaryotes and eukaryotes. Nevertheless, prokaryotic and eukaryotic kinases are not similar to each other from a structural point of view. In this study, we are mainly focused on the elucidation of DAGK isolated from BCG Tokyo 172 strain, a vaccine used in Taiwan. Try to find this experimental BCG diacylglycerol kinase within the structure of choice DAGK its recombinant gene, the E. coli bacteria into the expression. Based on the analyses using SDS-PAGE and circular dichroism (circula dichroism, CD) spectroscopy, the protein approximate molecular weight and its secondary structure, respectively, were established. DAGK contains 77.6% α-helix, 6.63% β-sheet, 6.43% turn and 10.66% Random coil. In addition, according to the amino acid sequence and using a computer software, the three-dimensional structure of DAGK was simulated. The two sets of structural information obtained from CD and computer simulation were compared and the results show that the secondary conformations of DAGK were believed to change hugely when the solvent polarity was increased. The results presented here suggest an important feature of DAGK and can be helpful in the study of new proteins with significant biological functions in addition to providing a sound basis for the design of future drug target molecules.

第一章 前言 1
1.1結核病與卡介苗介紹 1
1.2甘油二酯激酶(Diacylglycerol kinases, DAGKs/DGKs) 2
1.3原真核的DAGK介紹 4
1.4研究動機 8
第二章 實驗材料與方法 9
2.1實驗架構 9
2.2實驗材料 10
2.2.1質體與菌種 10
2.2.2實驗儀器與藥品 10
2.2.2.1儀器 10
2.2.2.2藥品 11
2.3實驗方法 12
2.3.1引子設計 12
2.3.2小量質體(plasmid DNA)之製備 12
2.3.3 DNA限制酶反應 13
2.3.4洋菜膠膠體電泳 13
2.3.5大腸桿菌E. coli BL21-RIL (pET28a-DAGK)蛋白質誘導 14
2.3.6親和性管柱製備及蛋白質純化 14
2.3.6.1 Protein 處理 14
2.3.6.2親和性管柱層析純化步驟 15
2.3.7 SDS聚丙烯醯胺膠體電泳法(SDS-PAGE) 17
2.3.8西方墨點法 17
2.3.9蛋白質再折疊與透析 19
2.3.10蛋白質定量(Bradford Assay) 19
2.3.11圓二色光譜(Circular Dichroism；CD) 20
2.3.12蛋白質3D結構分析 21
第三章 實驗結果 22
3.1 DNA限制酶反應 22
3.2 IPTG誘導蛋白質大量表現 22
3.3蛋白質純化與鑑定 23
3.4蛋白質再折疊與透析 24
3.5蛋白質二級結構 24
第四章 實驗討論 25
4.1 IPTG誘導蛋白質大量表現 25
4.2蛋白質純化 25
4.3蛋白質構型分析 26
第五章 結論與未來展望 27
第六章 文獻 28
附錄 44
附錄一 pET28a(+)質體 44
附錄二 DAGK蛋白質之胺基酸與其基因之DNA序列 45
附錄三 Diacylglycerol kinase的3D分子模擬圖示 47
附錄四 DAGK蛋白質3D模擬數據資料 48
附錄五 凝膠體電泳(SDS-PAGE) 49
附錄六 純化蛋白的buffer製備 52
附錄七 實驗室常用配方表 53附錄八 CDPro軟體操作 55

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