Rapid and simplified purification of recombinant adeno-associated virus.
Rapid and simplified purification of recombinant adeno-associated virus.
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DOI：
10.1016/j.jviromet.2012.04.004
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发表时间：
2012-08
影响因子：
3.1
通讯作者：
Gittes GK
中科院分区：
文献类型：
--
作者：
Guo P;El-Gohary Y;Prasadan K;Shiota C;Xiao X;Wiersch J;Paredes J;Tulachan S;Gittes GK
临床前基因治疗的体外和体内研究都需要高纯度的腺相关病毒（AAV）制剂。目前用于纯化AAV的方法需要使用CsCl或碘克沙醇梯度离心，或使用色谱法。这些方法可能是繁琐和昂贵的，需要高速梯度离心，或者对于色谱法，需要使用其他昂贵的设备。此外，这些方法都很耗时，病毒产量也不高。目前，除AAV血清型2外，没有商业纯化试剂盒可用。使用简化的方法纯化AAV，其病毒产量能够有效地用于成年和胚胎小鼠。该方法不需要超速梯度离心，也不需要层析。相反，聚乙二醇（PEG）/水两相分配用于从PEG 8000沉淀的病毒-蛋白质混合物中除去可溶性蛋白质。该方法快速获得高达95%的高质量纯化AAV回收率。整个纯化过程，包括HEK-293细胞转染，可以在一周内轻松完成，纯度似乎高于一轮CsCl梯度纯化后获得的纯度。
Preclinical gene therapy studies both in-vitro and in-vivo require high purity preparations of adeno-associated virus (AAV). Current methods for purification of AAV entail the use of centrifugation over either a CsCl or iodixanol gradient, or the use of chromatography. These methods can be cumbersome and expensive, necessitating ultrahigh speed gradient centrifugation or, for chromatography the use of other expensive equipment. In addition, these methods are time consuming, and the viral yield is not high. Currently no commercial purification kits are available for other than AAV serotype 2. A simplified method was used for the purification of AAV, with a viral yield that is able to be used effectively in adult and embryo mice. The method does not require ultrahigh speed gradient centrifugation nor chromatography. Instead, polyethylene glycol (PEG) / aqueous two-phase partitioning is used to remove soluble proteins from the PEG8000 precipitated virus-protein mixture. The procedure obtained rapidly up to 95% recovery of high quality purified AAV. The entire purification process, including HEK-293 cell transfection, can be completed readily within one week, with purity seemingly higher than that obtained after one round of CsCl gradient purification.
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影响因子：
12.4
作者：
Zincarelli, Carmela;Soltys, Stephen;Rabinowitz, Joseph E.
通讯作者：
Rabinowitz, Joseph E.
DOI：
10.1073/pnas.56.1.86
发表时间：
1966-01-01
影响因子：
11.1
作者：
ROSE, JA;HOGGAN, MD;SHATKIN, AJ
通讯作者：
SHATKIN, AJ
影响因子：
12.4
作者：
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通讯作者：
Henning, Phillip P.
影响因子：
7.7
作者：
Wang, Z;Zhu, T;Xiao, X
通讯作者：
Xiao, X
影响因子：
5.1
作者：
Zolotukhin, S;Byrne, BJ;Muzyczka, N
通讯作者：
Muzyczka, N