质粒pcDNA3.1-IL-24的制备和纯化

Preparation and purification of plasmid pcDNA3.1-IL-24

探索基因治疗用质粒pcDNA3.1-IL-24的规模化制备和纯化工艺。碱性裂解提取质粒后,以异丙醇和LiCl沉淀法去除大分子RNA。然后,使用DEAE Sepharose FF离子交换层析去除染色体DNA、小分子RNA、蛋白质等杂质。经本工艺纯化的质粒pcDNA3.1-IL-24浓度为727μg/mL,纯度为1.87,蛋白质含量为0.007μg/mL质粒DNA,未检测到RNA、抗生素残留。此工艺避免使用动物源性酶类及有毒试剂,有效去除质粒DNA制备过程中产生的杂质,可实现药剂水平质粒DNA的规模制备。

Large-scale preparation and purification of plasmid pcDNA3.1-IL-24 for gene therapy are explored in the paper.In the extracted plasmid with alkaline lysis method,the macromolecular RNA is removed,and then some other impurities like chromosomal DNA,micromolecular RNA and protein are get rid of with Sepharose FF ion exchange chromatography.The concentration,purity and protein content of the purified plasmid pcDNA3.1-IL-24 are 727 μg/mL,1.87 and 0.007 μg/mL plasmid DNA respectively.No any RNA or antibiotic are detected.The process avoids any animal-derived enzymes or toxic solvent to remove the impurities from plasmid DNA and can realize the large-scale preparation and purification of therapeutic-grade plasmid DNA.