Construction Of Recombinant Adenovirus Vector Co-expressing The E And M Gene Of Porcine Reproductive And Respiratory Syndrome Virus

porcine reproductive and respiratory syndrome(PRRSV) is the causative agent of PRRS,It causes an important disease in pigs characterized by reproductive failure in sows ,pneumonia and an increase in perinatal mortality in gilts .Previous study demonstrated that PRRSV has 8 structure proteins. The GP5 protein(EP)and the M protein(MP),which are major immun antigens of porcine reproductive and respiratory syndrome antigen,are the hotspot of researching recombinant vaccine against PRRSV.In this study,the recombinant adenovirus containing the E and M genes simultaneously was successfully obtained for the first time. To construct recombinant adenovirus,first,the ORF5 and ORF6 were amplified by RT-PCR , then linked them by FMDV 2A sequence obtained E+2A+M ,and then cloned into shuttle vector pAdTrack-CMV,being screened and obtained a recombinant shuttle plasmid of pAdTrack-CMV/E+2A+M ; Secondly,using pAdTrack-CMV/E+2A+M and the pAdEasy system by employing homologous recombination was efficiently constructed into E. coli strain BJ5183,bearing exogenous PRRSV genes of recombinant adenovirus;Third,the recombinant vector construct was cleaved with PacI ,and transfected into low passage 293 packaging cell line. The strain of recombinant adenovirus containing E and M gene from PRRSV strain was successfully obtained. This treatise providing a basis for the research of recombinant replication-defective adenovirus vaccine co-expressing the EP and MP of PRRSV .