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质粒DNA(pDNA)作为重要的基因治疗药物载体,其广泛应用受纯度和产量的限制。为了获得高纯度的pDNA,首先制备超大孔连续床晶胶基质,接枝二乙氨基乙基葡聚糖得到阴离子交换型晶胶介质;然后以pUC19质粒为例,将目标质粒转化至大肠杆菌,培养收集,碱液裂解和离心;最后用阴离子交换型晶胶介质从离心上清液中一步法层析分离pDNA。通过优化层析过程的pH值和洗脱条件,最终在pH值为6.6时,用0.5 mol/L的NaCl溶液洗脱,得到较高纯度的pDNA。整个分离过程中不使用动物源性酶,也不需常规分离中的高毒试剂,使获得pDNA的过程和产物更加安全。
Plasmid DNA (pDNA) as an important gene therapy drug carrier, its wide range of applications by the purity and yield restrictions. In order to obtain high-purity pDNA, an ultra-large-pore continuous bed gel matrix is prepared and grafted with diethylaminoethyl dextran to obtain an anion-exchange gellan gum medium; and then the pUC19 plasmid is taken as an example to transform the target plasmid into Escherichia coli, Culturing and collecting, lysis and centrifugation of lye; finally, pDNA is separated and purified by one-step chromatography from the supernatant with an anion exchange type crystal gelatin medium. By optimizing the pH value of the chromatographic process and the elution conditions, the final elution with 0.5 mol / L NaCl solution at a pH of 6.6 yielded higher purity pDNA. The entire separation process does not use animal-derived enzymes, nor the high toxicity of conventional separation reagents, the process of obtaining pDNA and products more secure.