CTAB 选择性沉淀和纯化质粒DNA 工艺的建立
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摘 要: 通过直接在大肠杆菌碱裂解上清中加入十六烷基三甲基溴化铵(CTAB), 优化CTAB 与质粒DNA 量的比例、质 粒DNA 选择性释放溶液的选择和TritonX-114 的使用, 建立了简单、易行的大规模质粒DNA 纯化工艺。纯化质粒DNA 的质量检测结果显示, CTAB 纯化的质粒DNA 无菌体RNA 污染, 菌体基因组DNA、内毒素和蛋白含量分别小于 100 ng/mg、50 EU/mg 和10 μg/mg 质粒DNA, OD260/OD280 比值介于1.75~1.85 之间, 超螺旋质粒DNA 的比例大于80%, 该工艺纯化的质粒DNA 能达到或接近FDA 规定的人用质粒DNA 的各项指标, 整个过程不使用动物源性酶和苯酚、氯 仿、无水乙醇等有毒或易燃、易爆试剂, 成本低廉, 工艺环保。
关键词: 十六烷基三甲基溴化铵, 质粒DNA, 纯化, 质量检测
Purification of Large-scale Plasmid DNAs by Selective Precipitation with Cetyltrimethylammonium Bromide
Abstract: Following Escherichia coli lysis with alkali, cetyltrimethylammonium bromide (CTAB) was directly titrated into the supernatant. An easy and feasible technology for plasmid purification was established with the optimized proportion between the quantity of CTAB and plasmid, combined with the specific solution for DNA release and TritonX-114 for endotoxin removal. Quality detection showed that the purified plasmid was free of contamination of host RNA. The host bacterial genomic DNA, endotoxin and bacterial protein were less than 10 μg, 50 EU and 10 μg per mg plasmids, respectively. The ratio of OD260/OD280 was between 1.75−1.85. Eighty percent of the prepared plasmids were presented in the supercoiled form. The plasmid purified with this technology can satisfy all criteria stipulated by FDA. The main advantages of the technology include the avoidance of animal-derived enzymes such as ribonucleases A, Proteinase K and toxic reagents like chloroform and phenol. In addition, the technology has low cost and no pollution.
Keywords: CTAB, plasmid, purification, quality detection
随着基因免疫和基因治疗研究的快速进展, 批量的动物试验、人体临床试验以及后续的应用需要制备大量高质量的质粒DNA, 而价格合理的生物制 品级重组质粒的规模制备一直是制约基因治疗和基 因免疫发展的瓶颈[1]。重组质粒的纯化主要涉及菌 体基因组DNA、RNA、蛋白质和内毒素的去除, 目 前使用的各种亲和层析法[2−4]、蛋白标签法[5]和金属 离子法[6−8]等在某些方面都存在成本高、步骤繁琐等 不足。本研究通过对CTAB 选择沉淀质粒DNA 方法 的优化、质粒DNA 的选择性释放和用TritonX-114 沉淀内毒素, 建立了低成本制备高质量质粒DNA 的 方法, 为以重组质粒为基础的核酸疫苗和基因药物 在临床上的推广应用奠定了基础。……
全文下载： http://img.dxycdn.com/trademd/upload/asset/meeting/2013/07/08/B1372750415.pdf
关键词: 十六烷基三甲基溴化铵, 质粒DNA, 纯化, 质量检测
Purification of Large-scale Plasmid DNAs by Selective Precipitation with Cetyltrimethylammonium Bromide
Abstract: Following Escherichia coli lysis with alkali, cetyltrimethylammonium bromide (CTAB) was directly titrated into the supernatant. An easy and feasible technology for plasmid purification was established with the optimized proportion between the quantity of CTAB and plasmid, combined with the specific solution for DNA release and TritonX-114 for endotoxin removal. Quality detection showed that the purified plasmid was free of contamination of host RNA. The host bacterial genomic DNA, endotoxin and bacterial protein were less than 10 μg, 50 EU and 10 μg per mg plasmids, respectively. The ratio of OD260/OD280 was between 1.75−1.85. Eighty percent of the prepared plasmids were presented in the supercoiled form. The plasmid purified with this technology can satisfy all criteria stipulated by FDA. The main advantages of the technology include the avoidance of animal-derived enzymes such as ribonucleases A, Proteinase K and toxic reagents like chloroform and phenol. In addition, the technology has low cost and no pollution.
Keywords: CTAB, plasmid, purification, quality detection
随着基因免疫和基因治疗研究的快速进展, 批量的动物试验、人体临床试验以及后续的应用需要制备大量高质量的质粒DNA, 而价格合理的生物制 品级重组质粒的规模制备一直是制约基因治疗和基 因免疫发展的瓶颈[1]。重组质粒的纯化主要涉及菌 体基因组DNA、RNA、蛋白质和内毒素的去除, 目 前使用的各种亲和层析法[2−4]、蛋白标签法[5]和金属 离子法[6−8]等在某些方面都存在成本高、步骤繁琐等 不足。本研究通过对CTAB 选择沉淀质粒DNA 方法 的优化、质粒DNA 的选择性释放和用TritonX-114 沉淀内毒素, 建立了低成本制备高质量质粒DNA 的 方法, 为以重组质粒为基础的核酸疫苗和基因药物 在临床上的推广应用奠定了基础。……
全文下载： http://img.dxycdn.com/trademd/upload/asset/meeting/2013/07/08/B1372750415.pdf
