Construction of Recombinant Adeno-associated Virus Vector Containing a siRNA Targeting 14-3-3ζ

[Objective] To construct the recombinant adeno-associated virus(AAV) vector containing siRNA targeting 14-3-3ζ.[Method] The siRNA was designed and synthesized,then inserted into plasmid psiRNA H1 promoter downstream.Western-Blot method was adopted to detect the inhibitory effects of siRNA.H1-si-ζ was amplified from psiRNA-14-3-3ζ,and then subcloned into plasmid pAAV-MCS-EGFP.Recombinant plasmid pAAV-14-3-3ζ-EGFP was co-transfected into AAV-293 cells with pAAV-RC and pHelper for recombinant AAV packaging.Expression of EGFP protein mediated by AAV was detected in the AAV-infected HT1080 cells under fluorescent microscope.[Result] The recombinant plasmid pAAV-14-3-3ζ-EGFP was verified by double digestion and sequence analysis.The detected green fluorescence indicated that recombinant AAV was successfully packaged and mediating gene expression.[Conclusion] The successfully packaged AAV-14-3-3ζ-EGFP provided references for further experiments of 14-3-3ζ analysis.