Objective: To construct a recombinant adeno-associated virus (AAV) shuttle vector expressing nerve growth factor beta (NGF-beta) gene.
Methods: By PCR amplification, the structural element of pAAV-multiple cloning site (MCS) and the functional element of pGenesil-1.1 were obtained and cloned into T-easy vector, respectively; the recombinant T-easy vectors were digested by restriction enzyme, then the target fragments were reclaimed and connected by DNA ligase, so the recombinant AAV shuttle vector pAAV-U6/CMV-enhanced green fluorescent protein (EGFP) containing U6 promoter and CMV promoter was obtained. The vector was transfected into 293 cells. The human Miapaca-2 cell line was cultured, and total RNA was extracted, then human NGF-beta gene was obtained by RT-PCR. T-easy-NGF-beta vector was constructed by cloning human NGF-beta gene into T-easy vector and identified by RT-PCR, digestion, and DNA sequencing. As NGF-beta gene was cloned into pAAV-U6/CMV-EGFP vector, the recombinant AAV shuttle vector expressing NGF-beta gene was obtained and identified by RT-PCR, digestion, and DNA sequencing.
Results: The bands of 800 bp and 4 250 bp were detected when pAAV-U6/CMV-EGFP was digested. The GFP was detected when pAAV-U6/CMV-EGFP was transfected into 293 cells. The bands of 736 bp and 3 015 bp were detected when T-easy-NGF-beta was digested; DNA sequencing result of T-easy-NGF-beta was fully consistent. The bands of 736 bp and 4 250 bp were detected when pAAV-U6/CMV-NGF-beta was digested. DNA sequencing result of pAAV-U6/ CMV-NGF-beta showed that sequences were completely correct.
Conclusion: The AAV shuttle vector pAAV-U6/CMV-NGF-beta is successfully constructed, providing experimental basis for investigation of the repair of spinal cord injury.