Li Gui-lin, Wang Ren-zhi, Wang Xin, Dou Wan-chen, Zhang Bo, Tian Shi-qiang, Ren Zu-yuan, Su Chang-bao
Department of Neurosurgery, Peking Union Medical College Hospital, Chinese Academy of Medical Science, Beijing 100730, China.
Zhonghua Yi Xue Za Zhi. 2003 Jul 25;83(14):1255-8.
To explore an efficient and simple way to product and purify helper free adeno-associated virus vectors.
Helper free rAAV system was transferred into HEK293T cells through phosphorated calcium method, thus producing rAAV, then the rAAV vector was purified through chloroform-PEG8000/NaCl-chloroform method and ultrafiltration. SDS-PAGE protein electrophoresis and Western blotting were used to detect the rAAV protein. The quantity of rAAV genome was determined through blot hybridization. HEK293T cells were cultured, rAAV was added, and fluorescence microscopy was used to count the amount of cells expressing green fluorescent protein so as to measure the transferring unit of rAAV.
rAAV2 was thus produced with a particle number of 2 x 10(13)/ml and a transferring unit of 5 x 10(11)/ml.
This method to produce and purify rAAV is efficient and simple, without need of any special equipment, and can be finished in a common laboratory.
探索一种高效、简便的生产和纯化无辅助腺相关病毒载体的方法。
通过磷酸钙法将无辅助rAAV系统转入HEK293T细胞，从而生产rAAV，然后通过氯仿-PEG8000/NaCl-氯仿法和超滤对rAAV载体进行纯化。采用SDS-PAGE蛋白电泳和Western印迹检测rAAV蛋白。通过斑点杂交测定rAAV基因组的数量。培养HEK293T细胞，加入rAAV，利用荧光显微镜计数表达绿色荧光蛋白的细胞数量，以测定rAAV的转导单位。
由此生产出rAAV2，颗粒数为2×10(13)/ml，转导单位为5×10(11)/ml。
这种生产和纯化rAAV的方法高效、简便，无需任何特殊设备，在普通实验室即可完成。
