Abstract:

Objective     To construct a vector of recombinant adeno-associated virus(AAV) containing the fusion gene hTRX-PR39, and explore the role of fusion gene hTRX-PR39 as a target gene in the treatment of cerebral ischemic disease. Methods       The constructed fusion gene hTRXPR39 was inserted into the EcoRIBamHI site of vector plasmid pSSCMV, and the vector of hTRX-PR39 recombinant AAV was constructed. The recombinant AAV stock was packaged. Renal embryo 293 cells were co-transfected with the recombinant AAV vector of plasmid pSSCMV/hTRX-PR39,packaging plasmid pAAV/Ad and helper adenovirus plasmid pFG140. Recombinant AAV was produced by homologous recombination of the 3 plasmids in renal embryo 293 cells and its titer was measured by quantitative dot blot hybridization. Results      The vector of hTRXPR39 recombinant AAV was successfully constructed. High titer of recombinant AAV was obtained by homologous recombination in renal embryo 293 cells(3.46×1012-3.46×1013PFU/mL). Conclusion        The recombinant AAV vector encoding the gene hTRX-PR39 was successfully constructed in this study by molecular cloning and in vitro recombination techniques, laying the foundation for further research into genetic therapy of cerebral ischemic disease.

Key words: Thioredoxin; Antimicrobial peptide PR39; Fusion gene; Recombinant adeno-associated virus