Construction And Identification Of Replication Deficient Adenovirus Recombinant Expressing G 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, the attachment G glycoprotein is one of the neutralization antigens, which can induce protective immunity against RSV infection, and a new type of vaccine candidate antigen. In order to investigate the protective immunity and its mechanism, we obtained the replication deficient adenovirus recombinant expressing the G protein by exploiting homologous recombination method.Method: According to the sequence of G gene in the GenBank (M17212), a pair of specific primers were designed and synthesized. G gene was amplified from RSV infected HEp-2 cells by RT-PCR and cloned into pGEM-T easy vector. After sequence analysis, G gene was subcloned into eukaryotic expressive vector pcDNA3.1 (+). The resulting recombinant plasmid pcDNA3.1(+)G was confirmed by the restriction endonuclease assay and transfected into COS-7 cells by Lipofectamin 2000. Then, the transcription and expression of G protein was identified by RT-PCR and Western blotting, respectively. The gene of the G protein was subcloned further into the adenovirus shuttle vector pShuttle-CMV. The resultant constructs were linearized with Pme I ,and transformed into the RecA~+ E.coli strain BJ5183 carrying the supercoiled adenoviral vector pAdEasy-1. The recombinant adenoviral constructs were cleaved with...