关键词: 腺相关病毒载体, S100A1, 转基因治疗

Abstract: AIM: To construct the recombinant adeno-associated virus (rAAV) vector expressing hS100A1 gene and to verify the correct recombination.METHODS: The hS100A1 plasmid (pUC57-Simple-S100A1) and AAV framework plasmid (pAAV-IRES-ZsGreen1) were cleaved by restriction endonuclease BamHⅠand EcoRⅠ. The target gene fragments were connected together to generate a recombinant plasmid pAAV-IRES-ZsGreen1-S100A1 that was transfected into E. coli DH5a. The plasmid was confirmed to be constructed as expectation by enzyme digestion and sequencing. The recombinant plasmid pAAV-IRES-ZsGreen1-S100A1 was co-transfected into AAV-293 cells with pHelper and pAAV-RC for packaging of recombinant AAV. The efficiency of rAAV packaging was monitored under fluorescent microscope and recombinant viral particles were harvested from infected AAV-293 cells. The virus titer was measured through infecting HEK-293 cells, and the recombinant rAAV-IRES- ZsGreen1-S100A1 was verified by PCR of the exogenous interest genes of S100A1.RESULTS: The recombinant plasmid pAAV-IRES-ZsGreen1-S100A1 was verified by double digestion and sequencing. Using the AAV helper-free system, ZsGreen1 expression could be observed under fluorescent microscope 72 hours after triple plasmid co-transfection and the system provided a high packing ratio of 95%-100%. The rAAV has a high purity and high titer of (2-3)×10⁷ TU/mL. The recombinant virus was confirmed by PCR of exogenous S100A1 genes.CONCLUSION: Recombinant rAAV-IRES-ZsGreen1-S100A1 was successfully constructed with a high virus titer, which may offer the basement of in vitro and in vivo experiments of hS100A1 for gene therapy of chronic heart failure.

Key words: Adeno-associated virus vector, S100A1, Transgenic therapy