| 阴离子交换晶胶层析分离质粒DNA |
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| 引用本文： | 郭延涛,沈绍传,贠军贤,姚克俭. 阴离子交换晶胶层析分离质粒DNA[J]. 生物工程学报, 2012, 28(8): 995-1001 |
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| 作者姓名： | 郭延涛 沈绍传 贠军贤 姚克俭 |
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| 作者单位： | 浙江工业大学化学工程与材料学院绿色合成技术国家重点实验室培育基地,浙江杭州,310032 |
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| 基金项目： | 国家自然科学基金 (Nos. 20876145, 21036005)，科技部中欧政府间国际合作项目 (No. 1017)，浙江省自然科学基金 (No. Y4080329) 资助。 |
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| 摘 要： | 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。
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| 关 键 词： | 超大孔连续床 离子交换 晶胶 质粒DNA |
| 收稿时间： | 2012-01-18 |
Chromatographic separation of plasmid DNA by anion-exchange cryogel |
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| Yantao Guo,Shaochuan Shen,Junxian Yun and Kejian Yao. Chromatographic separation of plasmid DNA by anion-exchange cryogel[J]. Chinese journal of biotechnology, 2012, 28(8): 995-1001 |
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| Authors: | Yantao Guo Shaochuan Shen Junxian Yun Kejian Yao |
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| Affiliation: | State Key Laboratory Breeding Base of Green Chemistry Synthesis Technology, College of Chemical Engineering and Materials Science, Zhejiang University of Technology, Hangzhou 310032, Zhejiang, China;State Key Laboratory Breeding Base of Green Chemistry Synthesis Technology, College of Chemical Engineering and Materials Science, Zhejiang University of Technology, Hangzhou 310032, Zhejiang, China;State Key Laboratory Breeding Base of Green Chemistry Synthesis Technology, College of Chemical Engineering and Materials Science, Zhejiang University of Technology, Hangzhou 310032, Zhejiang, China;State Key Laboratory Breeding Base of Green Chemistry Synthesis Technology, College of Chemical Engineering and Materials Science, Zhejiang University of Technology, Hangzhou 310032, Zhejiang, China |
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| Abstract: | Plasmid DNA(pDNA) is used as an important vector for gene therapy,and its wide application is restricted by the purity and yield.To obtain high-purity pDNA,a chromatographic method based on anion-exchange supermacroporous cryogel was explored.The anion-exchange cryogel was prepared by grafting diethylaminoethyl-dextran to the epoxide groups of polyacrylamide-based matrix and pUC19 plasmid was used as a target to test the method.The plasmid was transferred into Escherichia coli DH5α,cultivated,harvested and lysed.The obtained culture was centrifuged and the supernatant was used as the plasmid feedstock,which was loaded into the anion-exchange cryogel bed for chromatographic separation.By optimizing the pH of running buffer and the elution conditions,high-purity pDNA was obtained by elution with 0.5 mol/L sodium chloride solution at pH 6.6.Compared to the traditional methods for purification of pDNA,animal source enzymes and toxic reagents were not involved in the present separation process,ensuring the safety of both the purification operations and the obtained pDNA. |
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| Keywords: | supermacroporous monolith ion-exchange cryogel plasmid DNA |
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