臺灣博碩士論文加值系統

結核分枝桿菌(Mycobacterium tuberculosis)是造成結核病的主要病菌，根據世界衛生組織統計，全球每年平均感染肺結核菌的人數佔總人口的三分之一，是全球死亡率最高的傳染病，在2011年，約有九百萬的人口感染，其中約有一百萬的人口死於肺結核。台灣地區結核病發生率為每十萬人口中有6489人，屬於結核病中度盛行(median-prevalent)地區。以台灣地區而言，結核病發生率東部高於西部，南部高於北部，其中本土感染結核病與多重抗藥性結核病的發生率又以花蓮縣為最高。近年來多重抗藥性結核分枝桿菌的發生率逐漸升高，已經造成治療上的困難。目前立汎黴素 (Rifampicin) 和異菸鹼硫胼 (Isoniazid) 為臨床抗肺結核第一線藥物，研究發現常在抗立汎黴素結核菌rpoB 基因(the β subunit of RNA polymerase)發現點突變，而在抗異菸鹼硫胼結核菌katG 基因(Catalase-peroxidase-peroxynitritase T) 上發現點突變。為了解台灣東部多重抗藥性肺結核之分子特徵，本實驗收集台灣東部地區之臨床結核分枝桿菌228隻，分離出抗藥性結核分枝桿菌共67隻，並針對其抗藥性基因進行點突變分析。其結果發現抗藥性katG基因中的點突變，96% 屬於codon 315 (S315T)，主要是以單點突變為主；於抗藥性rpoB基因中的點突變，主要在codon 526 (22%)、531 (74%)或533 (20%)的位置，並於codon 548 (R548H) 的位置發現一個新的點突變。之後我們利用重組基因技術於Mycobacterium smegmatis 中表達rpoB 基因來證明點突變是否會造成Rifampicin抗性。結果顯示恥垢分枝桿Wild type菌株及vector control對Rifampicin的最低抑菌濃度經測試為2 μg/ml；S531L (positive control) 為16 μg/ml，而本篇所新發現的R548H則為4 μg/ml。結果顯示R548H的突變點會造成Rifampicin抗性。

Tuberculosis is caused by Mycobacterium tuberculosis. According to The World Health Organization (WHO) estimates that approximately one-third of the world’s population is infected with Mycobacterium tuberculosis (MTB), with an estimated nine million new cases reported and approximately one million people die from TB in 2011. In Taiwan, the Tuberculosis Incidence was 6489 per one hundred thousand belong to median-prevalent area in 2011 and almost detected in east or south of Taiwan, and most in Hualien. A potentially threat to TB control is the strains that can not be cured by standard anti-TB drug regimens. These cases will become multidrug-resistant TB (MDR-TB). Most of MDR-TB presented least two major anti-TB drugs [rifampicin (RIF) and isoniazid (INH)]. The rifampicin resistance TB results from a point mutation in the rpoB gene, encoding the  subunit of RNA polymerase and the isoniazid resistance TB results from a point mutation in the katG gene, encoding the catalase-peroxidase-peroxynitritase T. Among 67 clinical isolates from east of Taiwan were multi-drug resistant TB. The other 30 isolates were the isoniazid resistance TB and 50 isolates were the rifampicin resistance TB. Sequencing analysis of the katG gene showed no any new mutation difference, only occurred in codon 315, but the rpoB gene were founed a new point mutation (R548H). The other mutations occurred in the codon 526 (22%)、531 (74%) and 533 (20%). For this study, we constructed recombinant strains of Mycobacterium smegmatis carrying the rpoB gene with or without mutation. The rifampicin minimal inhibitory concentration results showed that the recombinant strains carrying the rpoB gene without mutation and vector control were 1 μg/ml, and the recombinant strain carrying S531L (positive control) and R548H were 8 μg/ml and 2 μg/ml, respectively。

目錄 I
附圖表目錄 III
中文摘要 IV
英文摘要(Abstract) V
第一章、前言 1
1.1結核分枝桿菌 （Mycobacterium tuberculosis）簡介 1
1.2恥垢分枝桿菌（Mycobacterium smegmatis）簡介 1
1.3肺結核傳染與病理 2
1.4 結核分枝桿菌檢驗鑑定 3
1.5結核病治療 5
1.6結核病抗藥性 7
1.7結核病流行病學 9
1.8研究目的 10
第二章、實驗材料 12
2.1 實驗使用菌株 12
2.2 實驗使用質體 13
2.3 限制酶 13
2.4 抗生素 14
2.5 引子 14
2.6 實驗藥品 14
2.7 培養基成分 15
2.8 質體DNA純化試劑 17
2.9 染色體純化試劑 17
第三章、實驗方法 18
3.1 染色體純化 18
3.2 質體純化 18
3.2.1 手工法 18
3.2.2 質體萃取套組法 19
3.3 限制酶切割 19
3.4 瓊脂膠體萃取DNA 20
3.5 DNA連接作用 20
3.6 轉型作用-熱休克法 20
3.6.1 熱休克法勝任細胞製備 20
3.6.2 熱休克法細菌轉型作用 21
3.7 轉型作用-電穿孔法 21
3.7.1 電穿孔法勝任細胞製備 21
3.7.2 電穿孔法細菌轉型作用 21
3.8 TA-載體選殖 22
3.9重組質體之建構 22
3.10 最低抑菌濃度測試 ( Minimal inhibitory concentration ) 25
第四章、實驗結果 26
4.1 臨床抗藥性結核分枝桿菌抗藥性基因之點突變分析 26
4.2 重組表達質體之建構及恥垢分枝桿菌勝任細胞條件優化 26
4.3 重組菌株對Rifampicin之最小抑菌濃度 27
第五章、討論 29
第六章、附圖表 31

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