Construction And Identification Of Replication Deficient Adenovirus Recombinant Expressing M Protein Of Human Respiratory Syncytial Virus

Objective Human respiratory syncytial virus(RSV) is the most important viral etiologic agent of pediatric respiratory disease worldwide.No effective vaccine is currently available for prophylaxis.The World Health Organization has affirmed RSV vaccines as one of the highest priority vaccine development projects.Among the encoded 11 viral proteins by RSV genome,matrix proteins play a central role in assembly of the virus.To investigate the role played by M protein in the protective immunity against RSV infection and its biological function,M gene of human respiratory syncytial virus(RSV) subgroup A is cloned,and replication deficient adenovirus recombinant expressing M protein is constructed.Methods According to the sequence of M gene,a pair of primers were designed and synthesized.M gene was amplified from RSV infected HEp-2 cells by using RT-PCR, and then cloned into pGEM-T easy vector.After sequence analysis,M gene was subcloned into eukaryotic expression vector pcDNA3.1(+).The resulting recombinant plasmid pcDNA3.1(+)/M was confirmed by restriction endonuclease assay,and then transfected into COS-7 cells by using Lipofectamine 2000.The expression of M protein was identified with Western blotting.The gene of the M protein was subcloned further into the adenovirus shuttle vector pShuttle-CMV.The resultant constructs were linearized with Pmeâ… ,and transformed into the E.coli strain BJ5183 carrying the supercoiled adenoviral vector pAdEasy-1.The recombinant adenoviral constructs were cleaved with Pacâ… to expose their inverted terminal repeats,and then transfected into 293 packaging cells to generate viruses.After the cytopathic effect(CPE ) appeared, the adenovirus recombinant was harvested and the expressed M protein was analyzed by Western blotting.Results DNA sequencing displayed no nonsense mutation in M gene.The transcription and expression of M gene in the replication deficient adenovirus recombinant were confirmed by Western blotting.The homologous recombination between the recombinant shuttle plasmid carrying the transgene and the adenovirus backbone plasmid was achieved in E.coli strain BJ5183.The resulting recombinant adenovirus plasmid was confirmed by the restriction endonuclease assay.After the recombinant adenovirus DNA was transfected into 293 cells line,the CPE of 293 cells was observed and the expressed M protein was detected by Western blotting.Conclusion The replication deficient adenovirus recombinant expressing M protein is obtained,which paves the way the investigation into the protective immunity against RSV infection and its mechanism.