Construction of the recombinant expression adenovirus vector carrying HIV-1 viral protein R gene and its expression in PC-3 cell line

Objective: To construct the recombinant adenovirus containing HIV-1 viral protein R(Vpr) gene,and investigate the expression of Vpr protein in PC-3 cells.Methods: The fragment of Vpr gene from expression vector pCI-neo-Vpr was cloned into the shuttle plasmid pAdTrack-CMV.With the backbone plasmid pAdEasy-1 and the Pme Ⅰ linearized shuttle plasmid pAdTrack-Vpr,the recombinant adenoviral plasmid was generated from the homologous recombination in the E.coli BJ5183.The adenoviruses were packaged in the AD293 cells by transecting the Pac Ⅰ linearized recombinant adenoviral plasmid,and were amplified by infecting AD293 cells,repetitively for 3-4 rounds.Then the viral titer was evaluated by green fluorescent protein(GFP) under fluorescence microscope.After infecting the PC-3 cells with the recombinant adenovirus at MOI of 1,5 and 10,the mRNA and protein expression of Vpr in PC-3 cells was detected by RT-PCR and Western blot,respectively.Results: Confirmed by the restriction enzyme digestion analysis,the recombinant adenovirus vector carrying Vpr was constructed successfully with the viral titer 3.0×108 efu/ml.And more than 80% PC-3 cells could be infected by the adenovirus at the MOI of 5.In addition,the expression of Vpr in PC-3 cells infected with AdEasy-GFP-Vpr was detectable by RT-PCR and Western blot.Conclusion: Recombinant adenoviruses with high titer and efficient infection of PC-3 cells could be obtained quickly and simply by using AdEasy system,and Vpr expression could be observed in PC-3 cells infected with AdEasy-GFP-Vpr.The results of this study lay the foundation for further studying on the role of Vpr protein in its anti-tumor activity and related signaling pathways.