| Adenoassociated virus type2 (AAV2) is one of the most promising gene transfer vectors in human clinical trials. In this study, we established the method for production and purification of AAV2-based vectors. HEK293 cells were transfected with pAAV/EGFP and PDG containing all AAV and Ad functions required for amplification and packaging of AAV vector plasmids. 72 hours later, cells were harvested and the rAAV /EGFP vectors contained in cells were purified using two methods: heparin column purification and CHCl3 purification. Then virus genome copies were determined by based-WPRE qRealtime-PCR, then viruses infected HEK293 with different M.O.I, the infectivities were observed. Compared CHCl3 with heparin column, CHCl3 purification method was more simple, economical and faster.The human IFNA1 cDNA coding BFNal was cloned by RT-PCR from blood lymphocytes, subcloned into pAAV vector and was named as pAAV/IFNAl. The nucleotide acid sequence of cloned IFNA1 was determined. rAAV/IFNAl virus wasgenerated and quantified according to the above methods. The pAAV/IFNAl then transfected HKE293 cells. The protein expression of the recombinant was assayed by western blotting. The result was found that one protein band with molecular mass of about 20kD was detected. It indicates that IFNa1 gene was correctly transcribed and translated in the infected cells. RAAV/IFNA1 virus infected AD38 cells containing HBV gene while using rAAV/EGFP virus as control. Sixteen hours post-infection, HBV contained in AD38 cells were released and AD38 culture medium was harvested every two hours and the HBV contained in AD38 cells were determined by realtime-PCR. The results showed that the amount of HBV contained in cells infected by rAAV/IFNA1 decreased gradually. It indicates AAV-mediated IFNa1 can be futher applied in therapy of HCC.This AAV production system can also deliver many interesting gene in animal test. It is a strong tool for gene therapy.
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