HIV-1型病毒蛋白R基因表达载体的构建及其在前列腺癌细胞系PC-3中的表达

Objective: To construct the recombinant adenovirus containing HIV1 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 RTPCR 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 3.0×10^8efu/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 PC3 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 antitumor activity and related signaling pathways.