CTAB法制备超螺旋质粒DNA  

Purification of Supercoiled Plasmid DNA by CTAB

作　　者：徐英黔[1,2] 吴蕾[2] 甘一如[2] 

机构地区：[1]辽宁科技大学化工学院,辽宁鞍山114051 [2]天津大学化工学院

出　　处：《过程工程学报》2007年第2期390-394,共5页The Chinese Journal of Process Engineering

基　　金：辽宁省'十一五'教育科学规划资助项目(编号2-14-10)

摘　　要：研究了基因治疗用载体质粒DNA的一种非色谱的大规模纯化方法—CTAB沉淀法,并对其进行了工艺优化．首先采用高纯过滤介质CelPure去除大量杂质,并利用CTAB浓度梯度溶液研究了不同形式DNA(超螺旋质粒DNA、切口质粒DNA、线性质粒DNA、染色体DNA)沉淀能力的差异,选择性地从蛋白质、RNA及其他形式DNA中提取超螺旋质粒DNA．针对含有pcDNA3的DH5α菌株,确定CTAB完全沉淀超螺旋质粒DNA的浓度为2．0g/L,由NaCl对不同沉淀的溶解能力,确定溶解沉淀的最优盐浓度为0．75 mol/L．用此方法得到的超螺旋质粒DNA的纯度达90％以上,收率为78．94％．RNA、蛋白质残量以及内毒素均符合药品质量标准要求．CTAB对质粒DNA的沉淀效果与质粒DNA的初始浓度有关,并且超螺旋质粒DNA浓度的降低与CTAB的添加量呈一定的线性关系．超螺旋质粒的分子量越小越不易被分离,并且结构特性比分子量在分离过程中更据主导作用．A new non-chromatographic method for the large-scale purification of pharmaceutical-grade plasmid by CTAB was developed. Meanwhile the purification process was optimized. Firstly, large amount of impurities in the crude plasmid mixture had been reduced dramatically by a high purity filter medimn-CelPure diatomite. Gradient concentrations of CTAB produced precipitation of plasmid variants. The selectivity mechanism is most likely based upon conformational differences among the several forms of DNA. The optimal concentration of CTAB to precipitate plasmid DNA is 2.0 g/L CTAB, and the optimal salt concentration for precipitating dissolution is 0.75 mol/L by using the plasmid pcDNA3 containing strain E. coli DH5α as a material. CTAB is effective for selective precipitation of plasmid DNA from proteins, RNA and the other relaxed and denatured forms of plasmid DNA. The final yield of supereoiled plasmid DNA is 78.94%, and the purity of the final concentrated solution is higher than 90% by this method. The result of plasmid DNA precipitation is related to initial plasmid concentration, and decline in concentration of supereoiled plasmid DNA is linear to CTAB increment. Supereoiled plasmid of molecular size is more important than purity. The molecular structure of Supereoiled plasmid is more important than molecular weight in the purification process.

关 键 词：质粒DNA 大规模纯化 溴化十六烷三甲基铵(CTAB) 

分 类 号：Q781[生物学—分子生物学]