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研究生:羅瀚倫
研究生(外文):HanLun Lo
論文名稱:分析與鑑定白色念珠菌MDR1抗藥基因之cis調控序列
論文名稱(外文):Analysis and identification of cis-acting regulatory elements of MDR1, a multidrug resistance gene in Candida albicans
指導教授:楊昀良
指導教授(外文):Yang Yun-Liang
學位類別:碩士
校院名稱:國立交通大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:58
中文關鍵詞:白色念珠菌MDR1基因刪減序列分析法
外文關鍵詞:Candida albicansMDR1 geneDeletion Analysis
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醫院的院內真菌感染已經變成了病患發病率和死亡率的一項重要因素了,尤其是患有免疫不全症候群、或進行輻射線治療、或是接受化學治療的病人特別容易感染。在現代醫學之中,醫生經常以抗真菌藥物來治療被真菌感染的病人。而隨著抗真菌藥物的廣泛使用,真菌的抗藥性已經變成了嚴重的問題。白色念珠菌是最常出現在免疫不全病人的伺機性病原菌。而最近研究顯示,將屬於Major Facilitator的MDR1基因,以基因破壞性突變(41)(gene disruption)和過量表現(7) (overexpression)來分析,發現此基因與抗藥性現象有所相關聯,但是關於此基因的分子調控機制仍然不清楚。為了近一步瞭解MDR1基因分子層面上的調控機制,我所著重的便是MDR1基因5端的基因表現的調控序列。本實驗所使用的序列分析方式是刪減序列分析法(Deletion Analysis),首先利用PCR方法針對此一區域將所欲得到的片段合成出來,構築在YEP363質體上,使成為MDR1 promoter-LacZ in-frame fusion,再去偵測此一突變株的β-galactosidase活性,如此分析所得到的數據,就可以推測出可能的順式調控序列所在。經過三次的刪減序列分析後,共預測出九個可能有順式調控序列的區間。在它們之中,-769 ~ -725的序列區間(以轉譯起始密碼子的第一個鹼基為+1)可能有負向的調控序列作用,當刪減此段序列時,相對於100 %有0.7倍的活性上升。另外由GCG比對的資料顯示,在-739 ~ -726的序列上有比對到七個不同種的順式調控序列可能的位置,針對此區域,於是利用定點突變法在質體pMDR763(-763 MDR1 promoter/YEP363 in-frame fusion)與pMDR1242 (-1242 MDR1 promoter/YEP363 in-frame fusion)進行一系列的突變分析,發現pMDR763的突變株都有活性上升的現象,確認在-739 ~ -726的序列區間確實存在有負向的調控因子。本實驗的研究結果將有助於我們更瞭解抗藥基因的調控機制,並且這些資料將可能應用於新藥物的開發。

Nosocomial fungal infection is an important cause of morbidity and mortality, especially to the patients having AIDS, undergoing radiation treatment, or accepting chemotherapy. In modern medicine, doctors often treat infected patients with antifungal drugs. As the antifungal drugs were widely used, drug resistance of fungi has became serious problems. Candida albicans is the most common opportunistic fungal pathogen from immunocomporomised patients. It had been reported that gene disruption(41) and overexpression(7) of certain major facilitator (MF) proteins encoded by the MDR1 gene are linked to the drug resistance phenomenon. But the molecular mechanisms controlling the expression was poorly understood. In order to understand the controlling mechanisms on molecular level, I have focused on the 5’ regulatory elements of MDR1. PCR methods were introduced for deletion analysis of this region. A YEP363 plasmids - MDR1 promoter-LacZ in-frame fusion has been constructed, to analyze the β-galactosidase activity of the mutants. Through activity assay, I have discovered nine regions that may have cis-acting regulatory elements. Among them, there may be negative regulatory elements in the -769 ~ -725 region (the first base of translation start codon as +1). When this region was deleted, it increased 0.7 time the relative activity. There are seven overlapping cis-acting regulatory elements in -739 ~ -726 region according to the database of GCG. Focused on this region, I haved used site-directed mutagenesis to proceed a series of mutational analysis on plasmids, pMDR763 (-763 MDR1 promoter/YEP363 in-frame fusion) and pMDR1242 (-1242 MDR1 promoter/YEP363 in-frame fusion). The phenomenon that pMDR763 mutants’ activity have raised reveals that the —739 ~ -726 region really contain negative regulators. The result of this research will improve our understanding of drug-resistant mechanisms on the level of gene regulation and these data may be applied to drug development.

緒論 ...................................................1
第一章 文獻回顧.........................................2
1-1.真菌的感染 .........................................2
1-1-1.院內感染 ......................................2
1-1-2.抗真菌藥物的作用機制 ..........................2
1-1-3.抗藥性 ........................................3
1-2.白色念珠菌 ........................................4
1-2-1.伺機性病原菌 ..................................4
1-2-2.多抗藥基因1 ...................................5
1-3.基因的上游序列 ....................................6
1-3-1.基因上游序列的調控 ............................6
1-3-2.上游序列的研究分析 ............................6
1-4.生物統計法 - 卡方檢定 ..............................7
第二章 材料、方法與步驟
2-1.材料 ..............................................8
2-1-1.菌株 ..........................................8
2-1-2.化學物品 ......................................8
2-1-3.儀器設備 .....................................10
2-1-4.藥品配製 .....................................10
2-1-4-1.培養基 ...................................10
2-1-4-2.緩衝溶液及溶劑 ...........................11
2-1-5.質體 .........................................12
2-1-6.引子 .........................................12
2-1-6-1.針對MDR1(SC5314)上游序列設計的引子........13
2-1-6-2.針對欲突變的區域(-769 ~ -725)設計的引子...13
2-2.實驗方法與步驟 ...................................15
2-2-1.質體DNA的製備 ................................15
2-2-2.聚合酶連鎖反應 ...............................15
2-2-3.限制酶反應 ...................................16
2-2-4.DNA連結反應 ..................................16
2-2-5.大腸桿菌勝任細胞的轉形 .......................16
2-2-5-1.大腸桿菌勝任細胞的製備 ...................16
2-2-5-2.勝任細胞的轉形 ...........................17
2-2-6.真菌的轉形 ...................................17
2-2-7.β-galatosidase活性分析 .......................17
2-2-7-1.蛋白質的製備 .............................17
2-2-7-2.蛋白質濃度的測定 .........................18
2-2-7-3.酵素活性的測定 ...........................18
2-2-7-4.酵素活性的計算 ...........................18
2-2-8.卡方檢定 .....................................19
2-2-8-1.計算方法和檢定原理 .......................19
2-2-9.真菌質體的取得 ...............................20
2-2-10.定點突變法 ..................................21
2-2-10-1.PCR反應 .................................21
2-2-10-2.DpnI酵素處理 ............................21
2-2-10-3.轉形及篩選突變質體DNA ...................21
第三章 結果
3-1.MDR1上游序列的分析...............................22
3-1-1.MDR1上游序列的取得及定序.....................22
3-1-2.MDR1上游序列的調控序列比對分析...............22
3-2.MDR1 promoter-LacZ的活性分析.....................22
3-2-1.構築MDR1 promoter-LacZ in-frame質體...........22
3-2-2.MDR1上游序列的刪減序列分析結果...............23
3-2-2-1.第一次刪減序列分析........................23
3-2-2-2.第二次刪減序列分析........................24
3-2-2-3.第三次刪減序列分析........................24
3-2-2-4.刪減序列分析資料的整合....................25
3-3.MDR1上游-763 ~ -725序列的定點突變分析............25
3-3-1.pMDR763的定點突變.............................25
3-3-2.pMDR1242的定點突變............................26
第四章 討論
4-1.MDR1上游序列的分析...............................27
4-1-1.MDR1上游序列的取得及定序.....................27
4-1-2.MDR1上游序列的調控序列比對分析...............27
4-2.MDR1 promoter-LacZ的活性分析.....................28
4-2-1.MDR1上游序列的刪減序列分析....................28
4-3.MDR1上游序列的定點突變分析.......................28
4-3-1.pMDR763的定點突變............................28
4-3-2.pMDR1242的定點突變...........................29
4-3-3.定點突變法的探討..............................30
4-3-4.選擇Saccharomyces cerevisiae作為表現MDR1上游序列
的菌株........................................30
4-3-5.β-galatosidase活性分析的探討..................31
4-4.未來展望與計劃.....................................32

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