Landázuri Natalia, Gupta Monique, Le Doux Joseph M
The Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology, Emory University, Atlanta, GA 30322, USA.
J Biotechnol. 2006 Oct 1;125(4):529-39. doi: 10.1016/j.jbiotec.2006.03.026. Epub 2006 May 2.
We have previously shown that the combined addition of Polybrene (PB) and chondroitin sulfate C (CSC) to retrovirus stocks leads to the formation of retrovirus-polymer complexes (i.e., flocs) that rapidly sediment onto cells, increases the efficiency of gene transfer, and can be used to rapidly concentrate and purify retrovirus stocks. The viruses remain associated with the polyelectrolyte complexes, however, which may complicate their use in downstream applications. In this study we determined if retrovirus could be flocculated using only one polymer (PB). We found that when retrovirus stocks were incubated with 320 microg/ml of PB, more than 70% of the viruses, and fewer than 0.3% of all other proteins, were pelleted by low-speed centrifugation. In contrast to retrovirus complexes formed with two polymers, retrovirus flocculated with PB disaggregated when they were resuspended in fresh medium. We conclude that flocculation of retroviruses with a single cationic polymer (PB) is a useful method for rapidly concentrating and purifying retroviruses, and may prove particularly useful when it is desirable to generate purified virus that is not part of a polymer complex.
我们之前已经表明，向逆转录病毒储备液中联合添加聚凝胺（PB）和硫酸软骨素C（CSC）会导致形成逆转录病毒-聚合物复合物（即絮凝物），这些复合物会迅速沉淀到细胞上，提高基因转移效率，并且可用于快速浓缩和纯化逆转录病毒储备液。然而，病毒仍与聚电解质复合物结合，这可能会使其在下游应用中的使用变得复杂。在本研究中，我们确定了逆转录病毒是否仅能用一种聚合物（PB）进行絮凝。我们发现，当逆转录病毒储备液与320微克/毫升的PB孵育时，超过70%的病毒以及少于0.3%的所有其他蛋白质通过低速离心沉淀下来。与用两种聚合物形成的逆转录病毒复合物不同，用PB絮凝的逆转录病毒在重新悬浮于新鲜培养基中时会解聚。我们得出结论，用单一阳离子聚合物（PB）对逆转录病毒进行絮凝是快速浓缩和纯化逆转录病毒的一种有用方法，并且当需要生成不属于聚合物复合物一部分的纯化病毒时，可能会特别有用。
