[[alternative]]Total protein expression induced by the gene-transformation of mono-substituted ruthenium complexes

碩士[[abstract]]以釕金屬錯合物作為抗癌藥物的相關研究，已成為近來熱門的研究方向。由文獻中證實，此類型錯合物對於大腸桿菌亦具有抑制效果 ，因此本實驗利用單一取代釕錯合物可鍵結 DNA之特性，以基因轉殖法研究釕錯合物鍵結BL21( DE3 )質體DNA對整體蛋白質表現影響，初步以[Ru(tpy)(bpy)Cl]Cl作為反應物(tpy=2,2'',2"-terpyridine; bpy=2,2''-bipyridyl)。利用限制酵素切割質體DNA使形狀由超螺旋狀轉變為直線狀，而導致電泳速率下降的特性，以Pvu Ⅱ限制酵素與BL21(DE3)之萃取質體作用，選出其中分子大小為3500 bp之質體 DNA作為與釕錯合物鍵結之反應物，並且以凝膠位移法證實釕錯合物與質體DNA的加成，加成物的電泳速率隨釕錯合物濃度增加而呈現階梯狀的降緩趨勢，此情形持續到釕錯合物濃度為10 μM才停止。Ru-DNA加成物以電穿孔基因轉殖方法送入 BL21(DE3)。由於所選用菌種原已具備了Amp r與lac-Z基因所限制，導致無法以藍白菌篩選後，由定序其質體DNA方法鑑定轉殖成敗，故以1.5 %洋菜膠電泳法觀測轉殖前後之核酸變化作為鑑定轉殖的方式。分子大小為14 kb之核酸產物會在 Ru-DNA作用下產生，而不含釕錯合物者則無。轉殖後所表現之整體蛋白質以超音波震盪法破菌取出，以SDS-PAGE進行比對。釕錯合物濃度由0 μM 增加至0.1 μM會導致蛋白質總表現量增加，然而當Ru錯合物濃度再增加至1,10 μM時，蛋白質總表現量反而下降。個別蛋白質差異則以28 kDa與40 kDa兩處蛋白質最為明顯。[[abstract]]Ruthenium complexes have attracted much attention in the development of anti-tumor drugs.Mono-substituted ruthenium complex,[Ru(tpy)(bpy)Cl]Cl(tpy = 2,2,2"-terpyridine;bpy = 2,2''-bipyridyl) have been known to bind with DNA resulting in the inhibition of the growth of Escherichia coli. The inhibition of Escherichia coli means total protein expression is altered. The relation between altered protein and the binding of [Ru(tpy)(bpy)Cl]Cl to DNA is what we would discuss in the article, and this is achieved by transferring the Ru-DNA adduct into Escherichia coli directly. In this article, the plasmid DNA with 2500 bp was extracted from BL21(DE3) and was binded with [Ru(tpy)(bpy)Cl]Cl. Binding condition is checked by gel mobility shift assay, and the Ru-DNA adduct showed decreasing electrophoresis mobility when [Ru(tpy)(bpy)Cl]Cl concentration increased from 0 to 10 μM. The Ru-DNA adduct were transferred into BL21(DE3) by electroporation method. Total protein induced by the gene-transformation of Ru-DNA adduct is attracted and analyzed by SDS-PAGE. The amount of total protein increased when [Ru(tpy)(bpy)Cl]Cl concentration is from 0 to 0.1 μM, but decreased from 0.1 to 10 μM. The apparent difference of individual protein expression is on the 28 kD and 40 kD gel band.[[tableofcontents]]中文摘要...................................................I Abstract..................................................II 謝誌.....................................................III 目錄...................................................... V 圖表目錄................................................VIII 符號與縮寫表...............................................X 前言.....................................................XII 第一章 簡介...............................................01 1-1 金屬錯合物於藥物發展上的應用........................01 1-2 釕錯合物的發展與應用................................05 1-3 核酸與蛋白質表現....................................09 1-4 凝膠位移法(Gel Mobility Shift Assay)................12 1-5 基因轉殖(Gene-transformation).......................18 1-6 抗生素與藍白篩選法..................................24 1-7 實驗目的與策略......................................29 第二章 材料與方法.........................................30 2-1 ㄧ般材料............................................30 2-2 儀器設備............................................33 2-3 藥品配置............................................34 2-4 實驗方法............................................37 2-4-1 實驗流程............................................37 2-4-2 E.coli BL21(DE3)培養................................37 2-4-3 E.coli BL21(DE3)質體DNA之純化與限制酵素確認.........38 2-4-4 凝膠位移法檢驗Ru-DNA加成物的形成....................43 2-4-5 電穿孔進行Ru-DNA加成物的基因轉殖....................43 2-4-6 基因轉殖之藍白篩選檢測..............................44 2-4-7 超音波震盪取得釕作用後BL21(DE3)粗萃取物.............48 2-4-8 洋菜膠電泳探討釕錯合物造成的核酸變化................49 2-4-9 SDS-PAGE探討釕錯合物濃度影響蛋白質表現..............49 第三章 結果與討論.........................................50 3-1 E.coli(DE3)質體DNA之取得............................50 3-2 萃取質體與pstI,PvuII限制酵素之作用結果..............51 3-3 以核酸電泳速率探討釕錯合物-DNA加成物(adduct)之形成..51 3-4 藍白篩選法與序列分析之結果..........................53 3-5 釕錯合物作用基因轉殖產生14 kb高分子量核酸產物.......54 3-6 E.coli BL21(DE3)整體蛋白質的取得與比較..............54 第四章 結論...............................................57 參考文獻..................................................58 附錄 附錄一 [Ru(tpy)(bpy)Cl]Cl分子結構.........................74 附錄二 pGEMT-T® Easy Vector, pGEM-3Z® Vector.............75 附錄三 QIAGEN Plasmid Midi Kit............................76 附錄四 Vector-NTI 8.0 操作步驟............................77 附錄五 SDS-PAGE 製膠流程..................................78 圖1-1 順鉑(cisplatin)之結構..............................1 圖1-2 cisplatin與DNA-GG-序列的交互連接 (cross-linking)...2 圖1-3 應用於臨床治療之金屬錯合物及結構...................3 圖1-4 進入臨床試驗階段的鉑系金屬錯合物...................4 圖1-5 (a)NAMI與NAMI-A (b)[RuII(η6-arene)(en)X]+.........6 圖1-6 釕錯合物與DNA鍵結模式..............................7 圖1-7 生命表現中心原則(central dogma)....................9 圖1-8 原核生命基因調控示意圖............................10 圖1-9 C5甲基化示意圖....................................11 圖1-10 洋菜膠(Agarose)分子結構...........................13 圖1-11 核酸電泳示意圖....................................14 圖1-12 EtBr分子鍵結核酸示意圖............................15 圖1-13 聚丙烯醯胺凝膠結構與組成..........................16 圖1-14 SDS作用蛋白質示意圖...............................17 圖1-15 氯化鈣基因轉殖法示意圖............................20 圖1-16 電穿孔基因轉殖法示意圖............................21 圖1-17 磷酸雙層構成細胞膜................................22 圖1-18 β-galactosidase作用分解X-gal示意圖...............24 圖1-19 藍白菌篩選法......................................25 圖1-20 T-A選殖法示意圖...................................26 圖1-21 DNA接合酶作用機制.................................28 圖2-1 洋菜膠電泳裝置圖..................................35 圖2-2 SDS-PAGE示意圖....................................36 圖2-3 單一菌落純化方法..................................37 圖2-4 限制酵素切割質體造成電泳速率下滑..................39 圖2-5 BC60 DNA序列示意圖................................45 表1 各濃度洋菜膠電泳適合分離的核酸大小範圍............14 表2 pstI與pvuII之辨識序列.............................40[[note]]學號: 693290214, 學年度: 9