超滤法去除重组腺相关病毒中内毒素的应用

为探讨超滤法在重组腺相关病毒精制中的可行性,采用不同截留分子量的超滤管对重组腺相关病毒样品rAAV9-Kal进行超滤处理.利用鲎试剂盒和qPCR法定量检测截留液和滤过液中的内毒素和病毒含量,评价超滤技术对样品中内毒素的去除效果.结果表明:在乙二胺四乙酸二钠和脱氧胆酸钠的预处理下,30 kD的超滤管对样品的回收率及内毒素去除率分别为0.912 2,0.870 6.因此,在单一超滤条件下,30 kD的超滤管是重组腺相关病毒样品超滤纯化的最佳选择.

Removal of Endotoxin From Recombinant Adeno-AssociatedVirus by Ultrafiltration

To explore the possibility of ultrafiltration technology used in recombinant adeno-associated virus purification, the recombinant adeno-associated virus sample termed rAAV9-Kal was first treated by different molecular weights cut off ultrafiltration devices, then the endotoxin and virus in the retentates and filtrates were assayed with quantitative tachypleus amebocyte lysate regents and real-time PCR respectively, and last the effects of ultrafiltration on endotoxin removal were evaluated. The results showed that the recovery rate and endotoxin removal rate of sample were 0.912 2 and 0.870 6 respectively, under the treatment of EDTA, DOC and 30 kD ultrafiltration devices. Therefore, the 30 kD should be the most suitable ultrafiltration device for sample ultrafiltration and endotoxin removal.