Construction and Detection of Recombinant Adeno-Associated Virus Co- Expressing PRRSV GP5, M Proteins and shRNA

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) is a severe threat to the swine industry and has caused heavy economic losses worldwide. The currently used inactivated and attenuated virus vaccines have several shortcomings, such as unsafety and low protection rate, so it is urgent to develop a new vaccine. To explore and develop a novel vaccine against PRRSV, a bi-functional recombinant adeno-associated virus, expressing ORF5 and ORF6 proteins as well as a short interfering RNA (shRNA) against ORF7, was constructed. The shRNA against ORF7 was inserted into the sequence forward of the U6 promoter of pAAV-U6-IRES-hrGFP, and ORF5 and ORF6 were cloned into the sequence after the CMV promoter. 293T cells that were co-transfected with this vector along with pAAV-RC and pHelper produced a recombinant adeno-associated virus. 293T cells transduced with this recombinant virus expressed GP5 and M proteins, and Marc145 cells transduced with this recombinant virus suppressed the replication of PRRSV. The infective titer of the reconstructed virus was 1.9 × 1010 v.gmL as measured by the dot blotting method, and GP5 and M proteins were detected by western blot. The successful construction of rAAV-shRNA-ORF5-6 paves the way for the development of novel bi-functional vaccines against PRRSV.