The construction and production of NT4-GFP-Ant recombinant adeno-associated virus

Objective To construct and produce the vector of NT4-GFP-Ant recombinant adeno-associated virus(AAV).Methods The resulting gene of NT4-GFP-Ant was inserted into the EcoR I-BamH I site of vector plasmid pSSHG to construct the vector of NT4-GFP-Ant recombinant AAV.The recombinant AAV viral sock was packaged.Renal embryo 293 cell was co-transfected with the rAAV vector of plasmid pSSHG/ NT4-GFP-Ant, packaging plasmid pAAV/Ad and helper adenoviruspasmid pFG140.The neuroblastoma cell was transfected with rAAVand the supernatant was transfered into 293 cell.Results The combinant viral stock vector of plasmid pSSHG/ NT4-GFP-Ant was constructed successfully.The results of dot blot showed that we had obtained the rAAV stocks of high titre(1.46×10~(12)PFU/ml).The fluorescence can be observed in the 293 cell with the fluores- cence microscope.Conclusion The viral stock of rAAV NT4-GFP-Ant was constructed and obtained successfully and the cell experiment confirmed that it can secret macromolecular transmembrane reporting proteins.We can further investigate the feasibility of the macromoleeular functional proteins passing through the blood-brain-barrer into brain and treating central nervous systerm diseases.