Construction And Identification Of Recombinant Adenovirus Or Baculovirus Expressing Structure Protein Of Human Respiratory Syncytial Virus

Objective:Human respiratory syncytial virus (RSV) is the most significant pathogen that causes serious lower respiratory tract infection in infants, old people and immunodeficiency patients, worldwide. RSV is an single, negative stranded RNA virus belonging to paramyxoviridae pneumovirus with genome encoding 11 protein. Attachment protein (G) and fusion protein (F) are the main neutralized antigens, meanwhile, matrix protein is the skelemin of RSV. Since the failure of formalin-inactivated vaccine, various kinds of RSV vaccines have been developed. No vaccines against RSV have been approved for use in humans due to safety and immunogenicity. Virus-liked particals (VLPs) have been widely used in the development of various kinds of vaccines and obtained a desired protective immunity. So far, there is no report of the immune effect and protective immunity of RSV VLPs, and the mechanism of RSV budding. To clarify the mechanism of RSV budding and large-scale prepare RSV VLPs, we construct and express RSV F, G, M by first generation replication deficient recombinan adenoviral vector (FGAd) or baculoviruse vector.Methods:For the construction of FGAd-Gsyn and FGAd-Msyn, the codon optimized full-length RSV M gene (Msyn) and G gene (Gsyn) were synthesized by Geneart (Regensburg, Germany). The Msyn and Gsyn were subcloned into the adenovirus shuttle vector pShuttle-CMV, respectively. After homologous recombination between pAdTrack and AdEasy-1 in BJ5183 cells, the pre-adenoviral plasmids encoding Msyn or Gsyn were produced. The resulting plasmids of pFGAd-Msyn and pFGAd-Gsyn were linearized by digestion with restriction enzyme Pacâ… and transfected into 293 packaging cells to generate FGAd-Msyn and FGAd-Gsyn. Finally, the expressed Msyn and Gsyn were identified by Western blot. For the construction of rBac-Fopt,rBac-Gopt and rBac-Mopt, the codon optimized full-length RSV F gene (Fopt),RSV M gene (Mopt) and G gene (Gopt) were synthesized by Geneart (Regensburg, Germany). Then, the recombinant baculovirus encoding Fopt,Mopt and Gopt were constructed and transfected into sf9 insect cells by Lipofectamine cellfectine reagent, respectively. Finally, the expressed Fopt,Mopt and Gopt protein were identified by Western blot.Results:At 7d, the CPE was observed after 293 cells transfected by pFGAd-Msyn or pFGAd-Gsyn, and the expressed Msyn and Gsyn were confirmed by Western blot. At 3d, the CPE of sf9 cells was observed after sf9 insect cells transfected by rBac-Fopt,rBac-Gopt and rBac-Mopt, and the expressed Mopt,Gopt and Fopt protein were detected by Western blot.Conclusion:We successfully obtained FGAd-Gsyn,FGAd-Msyn,rBac-Fopt,rBac-Gopt and rBac-Mopt.