Construction And Bioactivity Study Of The Recombinant Adenovirus Expressing GP5 And M Protein Of Prrsv

Porcine reproductive and respiratory syndrome(PRRS) is caused by the Porcine reproductive and respiratory syndrome virus (PRRSV)which causes reproductive disorders in pregnant sows and boars and respiratory problems in pigs of all ages.The protein GP5 encoded by the ORF5 relate the virus infection which induce the neutralizing antibodys and the main protective antigen; on the same time,the protein M is the most conservative in the structural proteins of PRRSV and have strong immunogenicity and induce the cellular immunity. So the protein GP5 and M are selected as the candidate genes in this study.In this study, the GP5 and M genes of PRRSV were amplifiered by RT-PCR and cloned into the eukaryotic expression vector pIRESneo which located the MCS and neor gene and obtained. The recombinant eukaryotic expression vector pIRES-GP5-M dihested by Mlu 1 and Bst1107 I and the ORF which contain the GP5 and M genes was cloned into the shuttle vector pacAd5 CMVK-NpA and the recombinant shuttle vecto rwas named pacAd5 CMVK-NpA-GP5-MThe shuttle vector plasmid pacAd5 CMVK-NpA-GP5-M and the the back-bone vector pacAd59.2-100 was linearized by PacI and then contransinfeced into the 293AD cells.The recombinant adenovirus rAd5v-GP5-M was packed after the homologous recombination in the cells.Subsequently the recombinant virus was detectived by PCR amplified. The results indiated that the GP5 and M gene were intergrated into the Adenovirus geneome. The titre determination tests indicated the TCID50 is 10-6.5/ml.Indirect immunofluorescence test and Western Blot assay indicated that the protein GP5 and M all were expressed in 293AD cells. This study established the fundation for the development of recombinant adenovirus vaccine against PRRSV.