Department of Surgery, Division of Pediatric Surgery, Children's Hospital of Pittsburgh, University of Pittsburgh, PA 15224, USA.
J Virol Methods. 2012 Aug;183(2):139-46. doi: 10.1016/j.jviromet.2012.04.004. Epub 2012 Apr 26.
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 HEK293 cell transfection, can be completed readily within one week, with purity seemingly higher than that obtained after one round of CsCl gradient purification.
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 AAV 的纯化方法包括使用 CsCl 或碘克沙醇梯度离心，或使用色谱法。这些方法可能很繁琐且昂贵，需要超速梯度离心，或者对于色谱法，需要使用其他昂贵的设备。此外，这些方法耗时且病毒产量不高。目前，除了 AAV 血清型 2 之外，没有商业纯化试剂盒。我们使用了一种简化的方法来纯化 AAV，其病毒产量足以有效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色谱法。相反，使用聚乙二醇（PEG）/水两相分配法从 PEG8000 沉淀的病毒-蛋白混合物中去除可溶性蛋白。该方法能够快速获得高达 95%的高质量纯化 AAV 的回收率。整个纯化过程，包括 HEK293 细胞转染，可以在一周内轻松完成，其纯度似乎高于 CsCl 梯度纯化一轮后的纯度。
