Construction Of Recombinant Adeno-associated Virus Vector Expressing The Rat GAP-43 Gene

Objective To construct the recombinant adeno-associated virus (rAAV) vector expressing the rat growth associated protein-43(rGAP-43) gene.Methods 1.The rGAP-43 open reading frame (ORF) cDNA was generated by RT-PCR using specific primers. This fragment was cloned into the pUCm-T vector and the result of sequence analysis was exact. Then the gene fragment was amplified by PCR through a pair of primers designed. 2. Meanwhile, the reporter gene—enhanced green fluorescence protein (EGFP) was digested with SalⅠ/BglⅡand cloned into plasmid AAV-MCS,to construct the recombinant AAV-EGFP plasmid .3. Then the GAP-43 gene fragment was digested with BamHⅠ/SalⅠand cloned into plasmid AAV-EGFP, to construct the recombinant AAV-EGFP-GAP-43 plasmid and that was verified by DNA sequencing and enzyme digestion. 4. The recombinant expression plasmid was co-transfect into the AAV-293 cells with pAAV -RC and pHelper by lipofectamine transfection method, then to observe the expression of EGFP under a fluorescence microscope and to package the rAAV and measure the infectivity titre of virus.Results 1.Both GAP-43 ORF gene fragment and the recombinant AAV-EGFP-GAP-43 plasmid were verified by enzyme digestion and DNA sequencing for correct reading frame and orientation. 2. GFP expression in co-transfected AAV-293 cells was observed and the titer of rAAV is 106particles/ml measured by that cells expressing green fluorescence were counted and compared with the total number of cells.Conclusion 1.Successful construction of the recombinant adeno-associated virus...