治疗用质粒DNA的纯化工艺  

A Purification Procedure for Therapeutic Plasmid DNA

作　　者：丛艳昭[1] 鲁承[2] 金宁一[1] 申镇维[1] 梁晓枫[2] 金洪涛[1] 白靓[1] 牟伟锋[1] 于长勇[1] 常巧呈[1] 齐楷[2] 金明杰[3] 

机构地区：[1]军事医学科学院军事兽医研究所,长春130062 [2]延边大学农学院动物医学系,龙井133400 [3]通化市人民医院,通化134001

出　　处：《中国生物制品学杂志》2008年第10期899-902,共4页Chinese Journal of Biologicals

基　　金："863"重大项目(2006AA02Z447).

摘　　要：目的探索适合大规模生产治疗用质粒DNA的纯化工艺。方法采用中空纤维柱收菌、碱裂解,再应用中空纤维柱浓缩质粒,经分子筛、亲和、离子交换等层析分离纯化质粒DNA,并应用凝胶电泳、PCR、核酸蛋白检测仪、BCA蛋白检测试剂盒和内毒素检测试剂盒对所纯化的质粒DNA进行全面检定。结果所获得的超螺旋质粒DNA达到样品总质粒的91.2%;质粒DNA总纯度为1.8;内毒素含量小于10EU/mg;几乎检测不到蛋白质残留,未检出基因组DNA残留。各项指标均符合有关质量标准。结论所采用的纯化工艺省时省力,制备的质粒DNA纯度高,适用于大规模生产治疗用质粒DNA。Objective To develop a purification procedure suitable for large-scale production of therapeutic plasmid DNA. Methods The culture of recombinant E. coli JM109 transformed with plasmid pVAXI-MEG-p24-4-1BBL-OX40L were harvested by hollow fiber column chromatography, cleaved with alkaline, then concentrated by hollow fiber column chromatography again and purified by molecular sieve, affinity and ion exchange chromatography. Perform overall control tests on the purified plasmid DNA by gel electrophoresis, PCR, nucleic protein calculator, BCA protein assay kit and endotoxin assay kit. Results The purified supercoiled plasmid DNA contained 91.2% of total plasmid and reached a purity（A260 / A280） of 1.8. The endotoxin content was less than 10 EU/mg. Little residual protein and no residual genome DNA were detected. All the quality indexes of purified plasmid DNA met the relevant requirements. Conclusion The developed purification procedure was time- and labor-saving, effective and suitable for the large-scale production of therapeutic plasmid DNA.

关 键 词：质粒DNA 纯化 中空纤维柱 层析 

分 类 号：R392.33[医药卫生—免疫学]