Purification and characterization of retrovirus vector particles by rate zonal ultracentrifugation
Purification and characterization of retrovirus vector particles by rate zonal ultracentrifugation
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DOI：
10.1016/j.jviromet.2005.10.030
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发表时间：
2006-04-01
影响因子：
3.1
通讯作者：
Kamen, Amine
中科院分区：
文献类型：
--
作者：
Segura, Maria de las Mercedes;Garnier, Alain;Kamen, Amine
蔗糖平衡密度超离心仍然是逆转录病毒纯化中最广泛使用的技术。然而，通过这种常规方法获得的纯化病毒制剂通常含有大量的污染细胞膜囊泡。此外，蔗糖溶液具有高粘性和高渗透性，这会损害逆转录病毒颗粒的完整性和功能。为了克服这些局限性，开发了一种替代纯化技术，使用速率区带超离心和碘克沙醇作为梯度介质。重组逆转录病毒颗粒通过在10%FBS存在下悬浮生长的293-GPG包装细胞产生。通过在10-30%连续碘克沙醇梯度上的速率区带沉降纯化浓缩的上清液。从梯度的中心级分中回收完整且有活性的病毒颗粒。通过使用这种策略，实现了高水平的纯化，如枯草杆菌蛋白酶处理研究所示，没有明显的细胞膜囊泡污染。经SDS-PAGE分析和分子排阻色谱分析，逆转录病毒制剂的纯度超过95%。根据负染电子显微镜检查，纯化的颗粒大小和形态均一。此外，使用这种纯化策略可以将由293-GPG包装细胞产生的大量缺陷型逆转录病毒颗粒与功能性逆转录病毒颗粒分离。(c)2005 Elsevier B. V.保留所有权利。
Sucrose equilibrium density ultracentrifugation remains the most widely used technique for retrovirus purification. However, purified virus preparations obtained by this routine method usually contain considerable amounts of contaminating cell membrane vesicles. In addition, sucrose solutions are highly viscous and hyperosmotic which jeopardizes the integrity and functionality of the retrovirus particle. In order to overcome these limitations, an alternative purification technique using rate zonal ultracentrifugation and iodixanol as gradient medium was developed. Recombinant retrovirus particles were produced by 293-GPG packaging cells grown in suspension in the presence of 10% FBS. Concentrated supernatants were purified by rate zonal sedimentation on a 10-30% continuous iodixanol gradient. Virus particles were recovered intact and active from the central fractions of the gradient. By using this strategy, high levels of purification were achieved, with no evident contamination with cell membrane vesicles as indicated by subtilisin treatment studies. The level of purity of the retrovirus preparation is over 95% as shown by SDS-PAGE analysis and size-exclusion chromatography. Purified particles appear homogenous in size and morphology according to negative stain electron microscopy. In addition, large amounts of defective retrovirus particles produced by 293-GPG packaging cells can be separated from functional retrovirus particles using this purification strategy. (c) 2005 Elsevier B.V. All rights reserved.