Construction and identification of recombinant lentiviral vector of AQP5 gene

Objective To construct the recombinant lentiviral vector of AQP5 gene.Methods AQP5 gene sequence was amplified as target gene in vitro.The cDNA was inserted into plasmid pWPTS-GFP which was linearized by restriction endonucleases MluⅠand SalⅠ.The recombinant plasmid was transformed into competent DH5α cells.The grown colonies were identified by colony PCR and DNA sequencing.Recombinant lentivector plasmids and the other helping plasmids were co-transfected into 293T cells by CaCl2,and cell culture supernatant was collected after 48 h.The virus supernatant was concentrated and titered in 293T cells.The infection efficiency of the constructed virus was determined in SGC7901 cells.AQP5 expression in SGC7901 cells was determined by Western blot.Results DNA sequencing and Western blot demonstrated that the lentivirus vector was constructed successfully.The constructed virus were obtained and infected SGC7901 cells.The titer of concentrated virus was 1×108 TU/ml.Conclusion Recombinant lentiu iral vector of AQP5 gene is successfully constructed.