Preparation And Detection Of Co-expressing PRRSV ShRNA And GP5, M Protein In Recombinant Adeno-associated Virus

Porcine reproductive and respiratory syndrome (PRRS), characterized by severe reproductivefailure in sows and respiratory diseases in young pigs, was caused by porcine reproductive andrespiratory syndrome virus (PRRSV） that is an enveloped, single-stranded positivesense RNA virus.PRRS spreads wildly and continues to be a major problem to the pork industry around the globe. It isapparent that the current molecular vaccines are not so effective in protecting against infections with thegenetically diverse field strains of PRRSV as the traditional vaccines. In addition, there is an ambiguousperiod for antibody production after vaccination, so PRRS is likely outbreak during this time when theswine is challenged by PRRSV. What’s more, vaccination may results in further crazy of PRRSV whenthe already infected pigs were inoculated. In order to prevent and control the spread of the PRRSV,purify environment, improve public health, a bi-functional vaccine based on recombinantadeno-associated virus was constructed. We achieved the following fulfillments:1) A short interferingRNA that was got after two SH lines annealed against ORF7of PRRSV was constructed topAAV-U6-IRES-hrGFP and the recombinant was named pAAV-U6-IRES-hrGFP-shRNA. ORF5andORF6genes of PRRSV were then constructed to pAAV-U6-IRES-hrGFP-shRNA and the recombinantwas named pAAV-U6-IRES-hrGFP-shRNA-ORF5-ORF6.2)293T cells were transduced bypAAV-U6-IRES-hrGFP-shRNA-ORF5-ORF6, pHelper and pRC at a ratio of1:1:1. Cells wereharvested after72hours-cultivation. Then the reconstructed virus, rAAV-shRNA-ORF5-ORF6, waspurified by chloroform-PEG method, and then was analyzed by SDS-PAGE, the result indicated the titerwas up to1.9×1010v.g./mL.3) The PRRSV supressing test showed that shRNA could suppress thereplication of PRRSV. Real-Time PCR assay indicated that the efficiency of shRNA that hindered thereplication of PRRSV was36.3%.4) The Western-blot assay indicated cells transduced byrAAV-shRNA-ORF5-ORF6could express GP5and M proteins of PRRSV. In general, we havedeveloped a novel bi-functional vaccine against PRRSV which paved the way for prevention andcontrol of PRRS.