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研究生: 黃慧茹
論文名稱: 第十七型脊髓小腦運動失調症:遺傳檢測及發展生化暨酵母菌的藥物篩檢模式
指導教授: 李桂楨
Lee, Guey-Jen
學位類別: 碩士
Master
系所名稱: 生命科學系
Department of Life Science
論文出版年: 2007
畢業學年度: 95
語文別: 中文
論文頁數: 84
中文關鍵詞: 脊髓小腦運動失調症多麩醯胺鏈藥物篩檢基因型分布
論文種類: 學術論文
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  • 脊髓小腦運動失調症(Spinocerebellar ataxia,簡稱SCA)是一群漸進性神經退化疾病,其大多是由於三或五核苷酸重複擴增所致,主要臨床症狀包括動作失調及發音障礙,主因是小腦退化及萎縮,目前計有28種之多。第十七型脊髓小腦運動失調症(SCA17)是位於染色體上6q27位置的TATA binding protein (TBP)基因CAG三核苷重複擴增所引起的神經退化性疾病。TBP基因所做出來的蛋白為細胞內重要的轉錄調節因子,而擴增的多麩醯胺(polyQ)可能影響TBP蛋白的功能。根據許多研究顯示帶有多麩醯胺擴增的蛋白會傾向於形成不溶解的聚集物(detergent-insoluble aggregate),進而造成神經細胞的功能異常、病變或死亡。針對TBP基因上CAG三核苷重複次數的族群研究顯示,在正常人族群中,TBP基因CAG三核苷重複次數大都為25~42,在家族性及偶發性的運動失調患者中,所發現的CAG三核苷擴增次數為43~66。本論文首先對長庚醫院神經內科所提供的正常人(197位)、運動失調症(ataxia)患者(13位)、帕金森氏症(Parkinson's disease)患者(103位)、失智症(dementia)患者(122位)、原發性顫抖症(essential tremor)患者(101位)及其他神經疾病患者(舞蹈症、肌張力異常症等,29位)進行SCA17 CAG三核苷重複擴增的分子檢測,結果並未發現任何擴增的等位基因,各族群中皆以36個重複序列的等位基因最常見。本研究的第二部份擬建立生化及酵母菌藥物檢測模式,在生化模式上先後製備了包含3~61個僅包含多麩醯胺鏈(GST-Qn)及包含多麩醯胺的不完整N端TBP (GST-tTBP-Qn)、完整N端TBP (GST-nTBPQn)、全長TBP (GST-fTBPQn)的GST融合蛋白,利用filter-trap assay及Western blot方法作為藥物測試,但可能因為TBP蛋白的溶解性及1C2抗體的適用性,控制組及待測組藥物皆無法有效抑制帶有多麩醯胺擴增蛋白的聚集。另外在酵母菌藥物篩檢模式上,製備了兩組不完整N端TBP (tTBP-Qn-EGFP)、完整N端TBP (nTBP -Qn-EGFP)綠螢光融合蛋白,表現於不同品系的酵母菌中,在半乳糖誘導融合蛋白表現模式下,可能原因為多麩醯胺的長度未達到影響酵母菌生長的長度,或半乳糖誘導效果不佳,表現多麩醯胺擴增綠螢光融合蛋白(tTBP-Q54-EGFP、nTBP-Q48-EGFP)的酵母菌,其生長與控制組(tTBP-Q20-EGFP、nTBP-Q20-EGFP)相較,並無差異。本篇論文提供發展生化及酵母菌藥物篩檢模式雖未達到預期結果,仍可提供未來相關研究的參考。

    Autosomal dominant spinocerebellar ataxias (SCAs) are a heterogeneous group of neurodegenerative disorders characterized by progressive cerebellar ataxia of gait and limbs variably associated with dementia, dysarthria, etc. More than 28 SCA types have been described. Among them, SCA type 17 (SCA17) is caused by an expanded polyglutamine (polyQ) in the TATA-box binding protein (TBP), a general transcription initiation factor. In addition to SCA17, neurodegenerative diseases including HD, SBMA, DRPLA and SCA types 1, 2, 3, 6, 7 are also caused by expansion of a CAG repeat tracts encoding expanded polyQ tracts. The expanded polyQ tract causes a conformational change in the polypeptide to promote misfolding and aggregation of the disease protein, leading to the death of neurons. Unrelated expanded alleles characterized in familial and sporadic ataxia patients are found ranging from 43~66 repeats as opposed to 25~42 repeats in the general population. We screened the SCA17 TBP gene in 197 normal controls and in patients with various neurodegenerative diseases: 13 ataxia, 103 Parkinson's disease, 122 dementia, 101 essential tremor, and 29 chorea and dystonia. The most common TBP allele contains 36 repeats and no expanded allele was found. Then biochemical and yeast assays were developed to screen effective chemical compounds which may inhibit polyglutamine protein aggregation. GST fused Qn, tTBP-Qn (truncated N-terminal TBP), nTBPQn (N-terminal TBP) and fTBPQn (full-length TBP) (n = 3 ~ 61) were overexpressed in E. coli BL21 cells and purified by affinity chromatography. After factor Xa cleavage to remove GST, the TBPQn proteins were used in drug screening by filter-trap assay and western blotting. However, neither controls (Congo red and trehalose) nor tested chemical compounds inhibit polyQ protein aggregation efficiently. This might be due to the low solubility of TBP and the suitability of 1C2 antibody. In addition, the expression of tTBPQ20/Q54-EGFP、nTBPQ20/Q48-EGFP under the control of the GAL1 promoter was induced by galactose in erg6 and W303 yeast strains. No growth inhibition was observed in yeast expressing full-length or N-terminal TBPQ54-EGFP proteins. This might be due to that low expression level was induced or the length of polyQ is not long enough to exhibit toxicity in yeast. Nevertheless, the study may provide information for future research design to develop assay to screen novel chemical inhibitors of aggregation.

    目錄......................................................I 圖目錄...................................................III 表目錄...................................................III 摘要.....................................................IV Abstract ................................................VI 壹、緒論 ..................................................1 一、神經退化性疾病........................................1 二、脊髓小腦運動失調症(SCA)................................1 三、第十七型脊髓小腦運動失調症(SCA17).......................3 四、藥物篩檢研究方向.......................................4 五、藥物篩檢研究模式系統...................................6 貳、研究目的................................................8 參、研究材料與方法 ..........................................9 一、血液樣品及化學合成化合物................................9 二、基因組DNA (genomic DNA)的萃取.........................9 三、聚合酶連鎖反應(PCR)及SCA17基因型分析(genotyping).......10 四、不完整N端、完整N端及全長TBP重組質體的構築...............11 (一) pGEX-5X-3-tTBP-Q3/Q61重組質體(不完整N端TBP).......11 (二) pGEX-5X-3m-nTBP-Q20/Q45/Q61重組質體(完整N端TBP)...11 (三) pGEX-5X-3m-fTBP-Q20/Q45/Q61重組質體(全長TBP)......12 (四) pGEX-5X-3m-TBP-Q20/Q45/Q60重組質體................13 (五) pYES2-tTBP-Qn-EGFP重組質體(不完整N端TBP)...........13 (六) pYES2-nTBP-Qn-EGFP重組質體(完整N端TBP).............14 五、重組質體的選殖 ..........................................14 (一)純化DNA片段........................................14 (二)接合反應...........................................15 (三)轉型勝任細胞(competent cell)之製備..................15 (四)細菌的轉型作用(transformation)......................16 (五)質體DNA之小量製備...................................17 (六)質體DNA的大量製備...................................17 六、酵母菌轉型勝任細胞的製備及轉型作用......................18 (一)轉型勝任細胞製備....................................18 (二)酵母菌的轉型作用....................................19 七、GST-TBP融合蛋白於細菌的表現、純化......................20 八、生化藥物篩檢模式的建立.................................20 九、酵母菌藥物檢測模式的建立...............................22 肆、結果...................................................23 一、SCA17 TBP基因CAG三核苷重複變異的遺傳分析...............23 二、重組質體的確認.......................................23 (一) pGEX-5X-3-tTBP-Q3/Q61重組質體....................23 (二) pGEX-5X-3m-nTBP-Q20/Q45/Q61重組質體..............24 (三) pGEX-5X-3m-fTBP-Q20/Q45/Q61重組質體..............25 (四) pGEX-5X-3-TBP-Q20/Q45/Q60重組質體................25 (五) pYES2-tTBP-Q20/Q54-EGFP重組質體..................26 (六) pYES2-nTBP- Q20/Q54 -EGFP重組質體................27 三、GST融合重組蛋白的表現.................................27 四、生化藥物篩檢模式的建立................................28 (一)不完整N端TBP蛋白..................................28 (二)完整N端TBP蛋白....................................29 (三)全長TBP蛋白.......................................30 (四)TBP蛋白...........................................31 五、酵母菌藥物檢測模式的建立...............................31 (一)tTBP-Qn-EGFP、nTBP-Qn-EGFP重組蛋白表現情形..........31 (二)酵母菌生長情形.....................................32 伍、討論..................................................33 一、建立台灣不同族群SCA17基因CAG重複之遺傳資料庫............33 二、生化藥物篩檢模式的建立................................33 (一)不完整N端TBP蛋白模式...............................33 (二)完整N端TBP蛋白模式.................................34 (三)全長TBP蛋白模式....................................35 三、酵母菌藥物篩檢模式建立................................36 (一)TBP-EGFP融合蛋白表現情形............................36 (二)酵母菌生長情形.....................................38 陸、參考文獻...............................................39 圖目錄 圖一、pGEX-5X-3-tTBP-Qn重組質體、融合蛋白及限制酶圖譜分析......50 圖二、pGEX-5X-3m-nTBP-Qn重組質體、融合蛋白及限制酶圖譜分析.....52 圖三、pGEX-5X-3m-fTBP-Qn重組質體、融合蛋白及限制酶圖譜分析.....54 圖四、pGEX-5X-3m-TBP-Qn重組質體、融合蛋白及限制酶圖譜分析......56 圖五、pYES2-tTBP-Qn-EGFP重組質體、融合蛋白及限制酶圖譜分析.....58 圖六、pYES2-nTBP-Qn-EGFP重組質體、融合蛋白及限制酶圖譜分析.....60 圖七、SCA17 TBP基因CAG三核苷酸重複次數分佈圖..................61 圖八、GST-fTBPQn融合蛋白的誘導與表現.........................62 圖九、純化後的GST-fTBPQn融合蛋白............................63 圖十、tTBP-Q3/Q61蛋白的filter trap藥物檢測試驗...............64 圖十一、nTBP-Q20/Q45/Q61蛋白的Coomassie blue染色及抗體染色分析65 圖十二、nTBP-Q20/Q45/Q61蛋白的western blot藥物檢測試驗.......66 圖十三、fTBP-Q20/Q45/Q61蛋白的Coomassie blue染色及 Western blot 藥物檢測試驗 ..........................................67 圖十四、流式細胞儀分析酵母菌(erg6品系)表現TBP-Qn-EGFP融合蛋白情形 .......................................................68 圖十五、流式細胞儀分析酵母菌(W303品系)表現TBP-Qn-EGFP融合蛋白情形 69 圖十六、不同品系酵母菌表現TBP-Qn-EGFP融合蛋白情形統計圖........70 圖十七、螢光顯微鏡觀察酵母菌表現TBP-Qn-EGFP融合蛋白情形........71 圖十八、erg 6品系TBP-Qn-EGFP融合蛋白對酵母菌生長情形的影響.....72 圖十九、W303品系TBP-Qn-EGFP融合蛋白對酵母菌生長情形的影響.....73 表目錄 表一、SCA17 TBP基因CAG三核苷重複各對偶基因的頻率分佈...........74

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