Abstract: To construct the recombinant adeno-associated virus vector carrying neuromedin U 2 receptor (NMU2R) short hairpin RNA (shRNA) for preparation of high-titer viruses, hairpin RNA was designed to target NMU2R rat mRNA, and was synthesized and cloned into pAAV-ZsGreen-shRNA plasmid which was double digested by BamH I and Hind III. The presence of the target sequence in the plasmid pAAV-ZsGreen-rNMU2R shRNA was confirmed by enzyme digestion, PCR and DNA sequencing analysis. And then the plasmid was transfected into AAV 293 cells. AAV 293 cells expression NMU2R shRNA were obtained and subsequently infected AAV packaging system to package the rAAV5-ZsGreen-rNMU2R shRNA. After purification, the functional and infectious virus was obtained and the titer of virus was 1×1012 vg/mL. Real-time PCR and western blot methods were used to detect the expression of NMU2R after infection with PC12 cells, and the empty viral vector pAAV-ZsGreen-shRNA was used as control. The results indicated that the expression of NMU2R mRNA and protein was decreased significantly after infection for 24 h and 48 h as compared with that of the control. In summary, a high-titer recombinant adeno-associated virus-5 vector carrying NMU2R shRNA has been constructed successfully.

Key words: neuromedin U 2 receptor, RNA interference, recombinant adeno-associated virus