Purification of human respiratory syncytial virus by ultracentrifugation in iodixanol density gradient.
Purification of human respiratory syncytial virus by ultracentrifugation in iodixanol density gradient.
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DOI：
10.1016/j.jviromet.2007.09.013
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发表时间：
2008-02
影响因子：
3.1
通讯作者：
Toms, G. L.
中科院分区：
文献类型：
--
作者：
Gias, E.;Nielsen, S. U.;Morgan, L. A. E.;Toms, G. L.
在蔗糖密度梯度中的超离心仍然是用于hRSV纯化的最常用的技术。然而，蔗糖的高粘度和高渗透性会对极不稳定的病毒造成损害，导致感染性丧失。为了克服这些局限性，开发了一种替代纯化技术，使用碘克沙醇作为梯度介质，加入MgSO 4作为稳定剂，EDTA在感染性试验前解聚病毒。通过在20-52%不连续碘克沙醇梯度上进行速率区带超离心纯化聚乙二醇浓缩病毒后，病毒颗粒在20-36%界面处形成条带。使用ELISA通过病毒融合糖蛋白含量确认病毒的存在。在20-52%连续梯度上通过浮力密度超离心进一步纯化后，在1.15-1.19 μ g/ml的密度区域回收病毒，这通过感染性滴度、病毒基因组和融合糖蛋白峰的重合来证实。回收率分析表明，使用碘克沙醇使病毒产量增加至69%。还发现碘克沙醇对感染性测定中使用的HeLa细胞无毒，消除了通过透析去除其下游的需要。
Ultracentrifugation in sucrose density gradient remains the most commonly used technique for hRSV purification. However, the high viscosity and hyper-osmotic property of sucrose can cause damage to the extremely labile virus leading to loss of infectivity. To overcome these limitations, an alternative purification technique was developed using iodixanol as gradient medium, incorporating MgSO4 as a stabilizing agent and EDTA to disaggregate the virus prior to infectivity assay. Virus particles were banded at the 20–36% interface after purification of polyethylene glycol-concentrated viruses by rate zonal ultracentrifugation on a 20–52% discontinuous iodixanol gradient. The presence of the virus was confirmed by viral fusion glycoprotein content using ELISA. After further purification by buoyant density ultracentrifugation on a 20–52% continuous gradient, the virus was recovered in the region of density 1.15–1.19 g/ml and this was confirmed by the coincidence of the infectivity titre, viral genome and fusion glycoprotein peaks. Analysis of recovery rates showed that the use of iodixanol increased the virus yield up to 69%. Iodixanol was also found to be non-toxic to HeLa cells used in infectivity assay, eliminating the need of its downstream removal by dialysis.
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影响因子：
3.8
作者：
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通讯作者：
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影响因子：
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作者：
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作者：
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