Construction of GFP reconbinant AAV- reporter virus and its expression in NIH 3T3 cell / 西安交通大学学报(医学版)

ABSTRACT

Objective To construct a recombinant AAV reporter virus expressing GFP and observe its expression in NIH 3T3 cell. Methods The resulting gene of GFP was subcloned into the site EcoRⅠand BamHⅠof vector pssHGCMV, and a recombinant adeno-associated virus (rAAV) vector pssHGCMV-GFP expressing GFP was constructed by using DNA recombinant technique. The 143 cells were transfected with the vector and the rAAV stock expressing GFP was packaged, produced and purified. The titre of the rAAV stock was measured by quantitative dot blot hybridization. NIH 3T3 cells were infected with the rAAV and the fluorescence was detected in alive cells to confirm successful infection and the expression of exogenous gene in NIH 3T3 cell line. Results A GFP-expressing rAAV vector VssCMV-GFP was successfully constructed. The titer of the VssCMV-GFP was 3.16?10~(12) particles/mL. GFP was initially expressed in the NIH 3T3 cells 48h following infected by VssCMV-GFP and subsequently it gradually increased. The green fluorescence in the cells was the most apparent 120h after infected. Conclusion VssCMV-GFP can infect the eukaryotic cells and GFP can be expressed in the alive cells.It is suggested that rAAV-mediated transfer of the gene to the eukaryotic cells can serve as a delivery system of gene therapy.