Expression Of FVIII Gene Carried By Lentivirus In Rat In Vitro

Objective To observe in vitro expression of blood coagulation factorⅧgene(hFⅧ) carried by Lentivirus by intramuscular injection.Methods Using calcium chloride method, recombined lentivirus was prepared by transinfecting carrier of lentivirus including hFⅧgene, packaging plasmid CMV△R8.74 and capsule plasmid VSV-G to toxin producing cell 293T. Hela cell was infected by the prepared recombined lentivirus, then the expression amount of hFⅧwas examined in supernatant of medium. The concentration of hFⅧantigen and antibody were examined after muscular injection to normal rat with the prepared recombined lentivirus. Genomic DNA getting from the injected tissue part was reacted in PCR machine.Results The hFⅧantigen was expressed as high as (0.7±0.1) ng/ml from supernatant after 48 hours of transduction. As high as (33.1±0.1) ng/ml level of hFⅧantigen was revealed in rat blood serum after muscular injection of the prepared recombined lentivirus (1×10~8IU/ml), and the duration was as long as 20 weeks. Serum hFⅧantibody was as high as (133.1±5.6) BU/ml and went on during experiment. No other main organs except the injected tissue part were observed existence of hFⅧgene.Conclusion The prepared recombined lentivirus expressed in the culture cell and could structure long time and permanent treating gene system in normal rats after muscular injection. It could be the theoretical and experimental basis of gene therapy of hemophilia A and other disease.