A novel method for purification of recombinant adeno-associated virus vectors on a large scale  

作　　者：WU Xiaobing DONG Xiaoyan WU Zhijian CAO Hui NIU Dongbin QU Jianguo WANG Hong HOU Yunde 

机构地区：[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

出　　处：《Chinese Science Bulletin》2001年第6期485-489,共5页

基　　金：the National High-technology Development Program (Grant No. Z20-05-02).

摘　　要：A novel method for recombinant adeno-associ-ated 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 2×1012 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.A novel method for recombinant adeno-associ-ated 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 2×1012 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.

关 键 词：RECOMBINANT ADENO-ASSOCIATED virus CHLOROFORM purifi- cation. 

分 类 号：R346[医药卫生—基础医学]