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

Porcine reproductive and respiratory syndrome(PRRS), commonly known as blue ear disease is caused by a porcine reproductive and respiratory syndrome virus(PRRSV), a highly contagious disease that can lead to sow miscarriage, premature birth, stillbirth, mummies and the decrease of viability and piglets severely respiratory symptoms.The genome of PRRSV is about 15kb and have eight open reading frames.GP2 protein, GP3 protein and GP4 protein are coded by ORF2,ORF3 and ORF4,that E protein,M protein and N protein are coded by ORF5,ORF6 and ORF7.E,M and N protein have a good immunogenicity,which make up major antigen of PRRSV. PRRSV is widely prevalent in the worldwide, causes economic losses and seriously harms the development of swine industry.The scholars have developed various vaccines to control the spread of PRRSV,adenovirus vector is received widely attention and applied for its own superiority.In this study, the recombinant adenovirus containing the E, M and N genes of PRRSV simultaneously was obtained for the first time. To construct the recombinant adenovirus, the E,M and N genes were amplified by RT-PCR and sequenced, linked them by FMDV 2A sequence which has self-cleavage function, obtained the sequence E-2A-M-2A-N, and then this sequence was cloned into shuttle vector pAdTrack-CMV,by PCR identification and restriction endonuclease map analysis,the pAdTrack-CMV/E-2A-M-2A-N was obtained;Secondly,the pAdTrack-CMV/E-2A-M-2A-N was transformed into the E.coli strain BJ5183 contained the pAdEasy-1, the homologous recombination was occured between them and gained recombinant adenovirus plasmid pAd/E-2A-M-2A-N containing exogenous genes of E-2A-M-2A-N.Thirdly, the recombinant vector was cleaved by PacI and transfected into HEK-293 packaging cell line, recombinant adenovirus containing E, M and N genes of PRRSV was successfully obtained. After subculture of the recombinant adenovirus, 4, 6, 8, 10 generations genomes of viruses were extracted and amplified by PCR, which confirmed that foreign genes have hereditary stability in recombinant adenovirus.Finally, plaque purification and TCID50 were to detect the toxic of virus.This provides a basis for the research of replication-defective adenovirus vaccine co-expressing the EP, MP and NP of PRRSV.