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標題: 使用水膠材質陰離子交換薄膜吸附分離質體核酸之研究
Plasmid DNA adsorption separation using hydrogel-based anion-exchange membranes
作者: 盧筱婷
Lu, Hsiao-Ting
關鍵字: plasmid DNA;質體核酸;hydrogel-based membrane;anion-exchange;水膠薄膜;陰離子交換
出版社: 化學工程學系所
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近年來因基因治療蓬勃發展與核酸疫苗技術的成熟,做為載體的質體核酸(plasmid DNA)不僅需求量大,在品質的要求上亦相對嚴格,故發展一套有效率的純化質體核酸技術為十分重要的課題。
本研究係探討商業水膠材質陰離子交換薄膜吸附帶負電生物分子之表現,進而應用於吸附分離大腸桿菌細胞鹼性溶離液之質體核酸,並探討其分離效果。首先,進行陰離子交換薄膜之批次吸附,所使用之吸附緩衝液為50 mM Tris-HCl,pH 8,於室溫下分別吸附質體核酸(6363 bp,約為4200 kDa)、牛血清蛋白(67 kDa)、寡核酸(50 bp,約為17 kDa)、及溶菌酶(14.4 kDa)。飽和吸附量排序為:寡核酸(9 μmol/ml)>牛血清蛋白(4 μmol/ml)>質體核酸(3×10-4 μmol/ml);溶菌酶因等電點為11,在吸附緩衝液下帶正電,故幾乎無吸附呈現,證明薄膜的不特定吸附低。
此外,貫穿曲線實驗結果比較,在貫穿點(c/c0= 0.1)時,質體核酸、牛血清蛋白、及寡核酸回收率約85-92%。而在吸附分離細胞鹼性溶離液中之質體核酸,所得到質體核酸之最適脫附條件為以2 M CaCl2 進行預沉澱處理,脫附於2 N NaCl,50 mM Tris-HCl,pH 8 中。其質體純度約可達100%,回收率約可達100 %。

The separation of plasmid DNA from cell lysate in downstream processing gains more and more attention. In most previous studies of some practical processes, such as traditional packed column. For reducing the mass transfer limitations which are usually encountered in the HPLC processes, hydrogel-based anion-exchange membranes were used to purify plasmid DNA in this work.
In batch adsorption process, the corresponding adsorption capacity of biomolecular could be sorted by Oligonucleotide (9 μmol/ml) > BSA (4 μmol/ml) > plasmid DNA (3×10-4 μmol/ml) in 50 mM Tris-HCl, pH 8 at room temperature.
In membrane chromatography process, the breakthrough curves of biomolecular were tested first. The order to achieve the breakthrough point (c/c0 = 0.1) from the earliest to the latest was: DNA > BSA > plasmid DNA. The recovery about 85-92 %. In cell lysate separation, the overall plasmid DNA purity about 100 %, recovery about 100 %.
其他識別: U0005-1808201013593100
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