以基因轉殖法研究單取代釕錯合物對整體蛋白質表現的影響

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

黃柏嘉(Po-Chia Huang)

指導教授 ： 鄭建中

淡江大學/理學院/生命科學研究所碩士班/碩士(2006年)

https://doi.org/10.6846/TKU.2006.00882

摘要

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。

關鍵字

釕金屬錯合物 ； 凝膠位移法 ； 基因轉殖法 ； 電穿孔法

並列摘要

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.

並列關鍵字

Ruthenium complex ； Gel Mobility Shift Assay ； Transformation ； Electroporation

參考文獻

2.Rosenberg, B.; Camp, L. V.; Krigas, T., Inhibition of

Cell Division in Escherichia coli by Electrolysis

Products from a Platinum Electrode. Nature 1965, 205,

Reviews 1999, 99, 2499-2510.

4.L.Miessler, G.; A.Tarr, D., Inorganic Chemistry. Second