Objective: To explore an efficient and simple way to product and purify helper free adeno-associated virus vectors.
Method: 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.
Results: rAAV2 was thus produced with a particle number of 2 x 10(13)/ml and a transferring unit of 5 x 10(11)/ml.
Conclusion: 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.