A novel method for purification of recombinant adenoassociated virus vectors on a large scale |
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Authors: | Xiaobing Wu Xiaoyan Dong Zhijian Wu Hui Cao Dongbin Niu Jianguo Qu Hong Wang Yunde Hou |
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Affiliation: | (1)State Key Laboratory for Molecular Virology and Genetic Engineering ,Beijing 100052 ,China; (2)Laboratory of Morphology, Institute of Virology, Chinese Academy of Preventive Medicine ,Beijing 100052 ,China |
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Abstract: | A novel method for recombinant adeno-associated virus (rAAV) purification on large scale is described. The method involves three steps, including chloroform treatment, PEG/NaCl precipitation and chloroform extraction. The whole procedure can be performed in four hours. Using this purification method, we can reproducibly obtain, from 4×109 of proviral cells cultured in roller bottles, purified rAAV-GFP stocks with titers of around 5×1013 particles/mL and purity greater than 95%. The infectious titers of the vector stocks were up to 2×10 TU/mL, thus particle-to-infectivity rate was about 25. Under an electronic microscope, most rAAV particles appeared full and a few were in intermediate form. Empty particles were rarely seen. The purified rAAV-GFP stocks have been successfully used in in vitro and in vivo transfection experiments. Therefore, this new method offers a simple, rapid and cost-effective way for large-scale rAAV purification. |
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Keywords: | recombinant adeno-associated virus chloroform purifi- cation |
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