摘要
The recombinant adeno-associated viral vector with human coagulation Factor Ⅸ minigene which wasregulated by CMV promoter was constructed. Largequantity of recombinant adeno-associated viral particles (rAAV/ hFⅨ) was prepared by the HSV/AAV hybrid helper virus method. Southern dot blot assay and QC-PCR indicated that the titer of the virus was 3.6×1012 v.g./mL. It demonstrated that this method can effectively overcome the hurdles of mass production of AAV vector. Followed by anintramuscular injection of viral vectors (7.5×1011 v.g./mouse) in the quadriceps femoris, an elevation of human Factor Ⅸexpression in the plasma of hemophilia B mice was detected (387 ng/mL) and persisted more than 12 weeks. The level of anti-virus antibody in plasma aligned with the Factor Ⅸexpression curve. The QC-PCR method is easier and moreaccurate than traditional dot hybridization fordetermination of the titer of recombinant adeno-associated virus. Moreover, there are no HSV particles existing inproduced AAV assayed by RT-PCR. AAV is the only virus that has been amplified from AAV-injected muscle by PCR.
The recombinant adeno-associated viral vector with human coagulation Factor Ⅸ minigene which wasregulated by CMV promoter was constructed. Largequantity of recombinant adeno-associated viral particles (rAAV/ hFⅨ) was prepared by the HSV/AAV hybrid helper virus method. Southern dot blot assay and QC-PCR indicated that the titer of the virus was 3.6×1012 v.g./mL. It demonstrated that this method can effectively overcome the hurdles of mass production of AAV vector. Followed by anintramuscular injection of viral vectors (7.5×1011 v.g./mouse) in the quadriceps femoris, an elevation of human Factor Ⅸexpression in the plasma of hemophilia B mice was detected (387 ng/mL) and persisted more than 12 weeks. The level of anti-virus antibody in plasma aligned with the Factor Ⅸexpression curve. The QC-PCR method is easier and moreaccurate than traditional dot hybridization fordetermination of the titer of recombinant adeno-associated virus. Moreover, there are no HSV particles existing inproduced AAV assayed by RT-PCR. AAV is the only virus that has been amplified from AAV-injected muscle by PCR.
基金
supported by the National Natural Science Foundation of China(Grant No.30100102).
