The Transduction And Expression Of ENOS Gene In HUVEC Mediated By RAAV8Vector

Objective:In this study, we will construct a novel virus vector recombinant adeno-associated virus contains fins-like tyrosine kinase-1(F1t-1) endothelial cell-specific promoter and Endothelial nitric oxide synthase(eNOS), and pack6type and8type recombinant adeno-associated virus. Through transduction cells in vitro, observe the purpose gene expression in Human Umbilical Vein Endothelial Cells.Methods:Part1Construction of pAAV-F1t-1-eNOSUsing pF1t-1-luc as a template, amplification the F1t-1promoter by PCR which contains Restriction sites, enzyme cut F1t-1fragment and existing plasmid of pAAV-MHC-TnT-GFP, construct a recombinant plasmid pAAV-F1t-1-GFP; Using pCMV6-XL6-eNOS as a template amplification the eNOS gene by PCR which contains restriction sites, cut eNOS fragment and plasmid of pAAV-F1t-1-GFP, construct a recombinant plasmid pAAV-F1t-1-eNOS.Part2Functional verification of F1t-1promoter and eNOS expression activity.Transfect293cells and HUVEC with pAAV-F1t-1-GFP and pAAV-D(+)-CMV-GFP, observe GFP expression with fluorescence microscope after24h. Transfect293cells with pAAV-F1t-1-eNOS, detect eNOS expression by Western Blot after.Part3Construction of recombinant virus vector of rAAV-F1t-1-eNOSThe rAAV6or rAAV8vectors were constructed by triple transfection methods in293cells. Using the pAAV-F1t-1-eNOS, pXX8�'�pXX6contains Rep and Cap gene, and pHelper. The virus titer of rAAV6-F1t-1-eNOS and rAAV8-F1t-1-eNOS were measured by Real-Time PCR.The virus purity of rAAV6and rAAV8were observed by SDS-PAGE.Part4Detect the expression of rAAV-F1t-1-eNOSInfect Human Umbilical Vein Endothelial Cells with rAAV-F1t-1-eNOS, collect cells after48h, detect the expression of eNOS with Western Blot.Results:1.Successfully construct the packaging plasmid of pAAV-F1t-1-eNOS2.The plasmids of pAAV-F1t-1-GFP、pAAV-F1-1-eNOS can be expressed in293cells.3.Successfully pack the recombinant virus rAAV6-F1t-1-eNOS and rAAV8-F1-1-eNOS,and the titre were3.14x1011v·g·/ml and1.12×1012 v.g./ml.4.The recombinant-virus rAAV-Flt-1-eNOS can normal expresse in the UVEC.Conclusion:LrAAV6-Flt-1-eNOS and rAAV8-Flt-1-eNOS can induced expression of exogenous gene in normal cells, F1t-1promoter can drive the specific expression of eNOS gene in HUVEC, the expression of rAAV6is superior to rAAV8in vitro.2.The successfully constructed of rAAV8-Flt-1-eNOS in the study provides a new way to achieve the targeted gene therapy for vascular endothelial cells with recombinant AAV vectors.