Construction And Identification Of Replication-defective Recombinant Adenoviruses Which Co-express The Fusion Protein Of RSV And EGFP

Objective: Respiratory syncytial virus (RSV) is an important viral pathogen that causes severe lower respiratory tract illness in infants and young children. This virus is distributed worldwidely. The mechanism of host defense against RSV infection remains unknown. Currentl, no licensed vaccine is available. The fusion glycoprotein (F) and attachment protein (G) are the only two neutralizing antigens of RSV. Due to the diversity of G protein, there are two antigenic subgroups, A and B, and subgroup A is more prevalent in China. Because of its structural and infective characteristic, we intend to combine the replication-defective adenoviral recombinant and DNA vaccine expressing both G and F glycoprotein of RSV to prevent RSV infection. The advantages of the F and G expressed in combination are as follows: they can induce a balanced immune response similar to live attenuated RSV vaccine; the transfection and the expression efficiency of foreign genes will be improved; a more efficient vaccination with less pain and reduced expense can be achived because two kinds of neutralizing antibodies will be produced by one shot of inoculation.At present, the coexpressive strategy are mainly used in gene therapy, or used to express the subunit of heterogenous polyprotein, but few reports have come from vaccine research. American BD Bioscience Clontech Company has commercialized eukaryotic vector pIRES, yet there is still no adenoviral vector system for bicistronic coexpression. We tried to utilize internal ribosome entry site (IRES) to construct adenoviral recombinant that can be used for bicistronic expression.Methods: According to the sequence of IRES, a pair of specific primers was disigned and synthesized. The commercial eukaryotic co-expressive vector pIRES was used to amplify IRES and its regulative sequences of IVS. Then, both IRES and IVS were cloned into T vector and the recombinant T vector pGEMT-IRES (IRES was inserted between MCSA and MCSB) was constructed. Based on the sequence of MCSA and MCSB, F gene and enhanced green fluorescence protein (EGFP) were amplified and cloned into pGEMT-IRES to construct...