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

Objective: Human Respiratory syncytial virus (RSV) is the most important cause of viral lower respiratory tract illness in infants and children worldwide. The mechanism of host immune protection against RSV infection remains unknown. So far no practical method for the prevention of its infection has been available. The fusion F glycoprotein and attachment G glycoprotein are the only two neutralization antigens of RSV. There are two antigenic subtypes, A and B, because of the diversity of G protein. F glycoprotein has long been recognized as a major vaccine candidate as it is an important target antigen for virus-specific cytotoxic T lymphocytes (CTL) and neutralizing antibodies. Furthermore, it is highly conserved among the two antigenic groups of RSV. In order to investigate the protective immunity and its mechanism, we obtained the replication deficient recombinant adenovirus expressing the F protein by exploiting homologous recombination method.Methods: According to the sequence of F gene, a pair of primers were designed and synthesized. The F gene was obtained by RT-PCR. Then the F gene was cloned into pGEM-3zf vector. After sequence analysis, F gene was subcloned into eukaryotic expressive vector pcDNA3.1(+). The resulting recombinant plasmid of pcDNA3.1 (+)/F was confirmed by restriction endonuclease assay, and the expression of F protein was identified with Western blot assay. The F gene was subcloned further into the adenovirus shuttle vector pShuttle-CMV. The resultant constructs were linearized with Pme I and transformed into RecA~+ E.coli strain BJ5183 carrying the supercoiled...