Recombinantadeno-associated virus (rAAV) is a developed vector. The rAAV is used to establish animal models of gene over-expression or knock down and as the tool of gene therapy. We use the three plasmids assay to package the rAAV which carries the EGFP gene in HEK293 cells. Through a series of experiments, we establish the method of rAAV purification. In this method, we lyse the cells using sodium deoxycholate, then remove the hybridproteins by adding high concentration NaCl, and purify the rAAV through heparin affinity column. After concentration by ultrafiltration, the rAAV titer can reach to more than 10¹³ gene copys /mL. The results of different multiplicity of infection (MOI) and time using rAAV to infect HEK 293 cell show that the best condition of infection is MOI=10⁶ and 3 days. After 3 weeks of injecting rAAV to substantia nigra pars compacta of rat midbrain, there are EGFP expressed in the dopaminergic neurons. We have established a method to prepare small amount rAAV in the lab, which can be used in vitro and in vivo.

recombinant adeno-associated viral  /  viral packaging  /  viral purification