Construction and identification of the recombinant lentiviral vector expressing human neurotrophin-3 gene

Objective: To construct a recombinant lentiviral vector expressing human neurotrophin-3(hNT3).Methods: pGC-E1-hNT3-EGFP plasmid was constructed by double restriction enzyme digestion and ligation,and then the plasmid was transformed into E.coli DH5α.The correct hNT3 gene was confirmed with PCR,endoenzyme digestion,sequencing analysis and contrast.The plasmid of pGC-E1-hNT3-EGFP vector was cotransfected together with lentivirus-packaging plasmid pHelper 1.0 and pHelper 2.0 into 293T packaging cells by Lipofectamine 2000 mediation.The newly constructed recombinant lentivirus and the titer of virus were confirmed with real-time quantitative PCR.Results: The 512 bp DNA sequence showed that the cloned hNT3 gene sequence was the same as that of the published sequence in GeneBank.The evidence of endonuclease digestion,DNA sequencing and PCR analysis confirmed that hNT3 gene was correctly inserted into the Ientiviral vector.and the titer of virus was 5 × 107 TU/L.Conclusion: The recombinant lentivirus expressing neurotrophin-3 gene are successfully constructed and effectively expressed in 293T cells,which probably can provide a reliable tool for genetic transfection in further experimental researches of nerve injury repair.