Construction And Immunogenicity Of PRRSV-1 VLPs

This study combined with the pathogenic mechanism?disease prevention and control and virus gene function research of porcine reproductive and respiratory syndrome virus,using PRRSV-1 GP5 protein as the main immunogen,using Bac-to-Bac~?baculovirus–insect cell expression system,through four different protein expression strategies to produce PRRSV-1virus-like particles which consisting of GP5 and M proteins,then analyzed their immunogenicity through the nasal mucosal immune pathway.PRRSV LV stains genome which preserved in our laboratory as a template.PRRSV-1 GP5and M genes were obtained by RT-PCR.Using bioinformatics and corresponding software to analyzed the evolutionary relationship and structure of the two gene and the GP5or M protein.At the same time amplified porcine IFN-?1 gene from porcine pulmonary alveolar macrophages,and fusion with the PRRSV-1 M gene,the result of analysis the signal peptide and transmembrane region show that IFN-?1 fusion may not affect the expression of M protein.The GP5 or M gene was cloned into the baculovirus shuttle vector pFastBac HTA/C or pFastBac Dual,respectively.Through the heat shock method made the recombinant shuttle vector into the DH10~?Bac E.coli competent cells;Blue-white spot screening was used to identify colonies containing recombinant bacmids rB-GP5?rB-M or rBD-GP5-M;The recombinant bacmid was transfected into SF9 cells using cationic liposome transfection method to rescue recombinant baculovirus which carrying GP5 and/or M genes.Recombinant baculovirus were expanded to the second generation.The recombinant baculovirus titer was determined by enzyme-linked immunospot assay.Western blot and indirect immunofluorescence were used to detect the expression of GP5 and M proteins.The expression of GP5 and M protein was detected at different time after the SF9 cell infected with the recombinant baculovirues in order to determine the best collection time of virus-like particles;A large number of PRRSV VLPs were prepared using a serum-free expression system;Cell culture supernatants were collected then ultracentrifugation using sucrose cushions and sucrose density gradient centrifugation to purified the VLPs;After purified,Western blot was used to identify the presence of GP5 and M.The morphology of the collected VLPs was observed by transmission electron microscopy.Finally,through intranasal immunization the BABL/C mice,the humoral immunity,cellular immunity,and mucosal immunity were tested.Analyzed the immunogenicity of VLPs and the VLPs-A5 complexes.The results showed that the strategy of co-expression of GP5 and M protein using pFastBac Dual vector to construct the recombinant bacmids rBD-GP5-M,packaging and save the recombinant baculovirus rBDV-GP5-M,infection SF9 cells in serum-free suspension culture can successfully packaged the VLPs with similar morphological structure of PRRSV-1.The purified VLPs as immunogen,immunized mice through the nasal immunization.In the experiment the weight of mice keep a steady upward trend,and did not cause excessive stress in mice;VLPs alone and VLPs+A5 could induce mice local sIgA secretion increased by nasal immunization,neutralizing antibody level of IgG increased.The immune response is mainly humoral immunity based on B cell immune response.The A5 delivery system allows the VLPs to release slowly,keeping the body's antibody level in a continuous rising state,providing a long-term immune response.The results indicate that PRRSV-1 VLPs have the potential to be developed as a safe and effective new type of mucosal vaccine,and the A5 delivery system can enhance the mucosal immune response.