Conatruction Of Recombinant Baculovirus Expressing ORF2 Gene Of Porcine Parvovirus 4 And Study Of Its Imumunogenicity

Porcine parvovirus 4 (PPV4) is a new pathogen discovered in recent years, it mainly cause porcine respiratory and reproductive disorders. The current research of PPV4 largely limited to its epidemiology and pathology, no suitable cell line in vitro have been found for the proliferation of this virus.Based on above, this study used baculovirus as vector to construct a recombinant baculovirus expressing the main structural gene of PPV4 ORF2, and studied the immunogenicity of it. The detailed contents are as follows:1. Prokaryotic expression of PPV4 VP gene and its purificationAccording to the antigen epitopes analysis of PPV4 VP, it was cut into 304-500aa, the fragment was cloned into pET28a prokaryotic expression vector after digested by BamHI and Hindlll to construct recombinant plasmid pET-28a-VP, induced the expression of the protein and purified it, coated ELIAS A plate with purified protein, determined the best concentration for packaging by phalanx titration to provide a detection method for the evaluation of immune effect of later experiments in mice.2. The construction of recombinant baculovirus expressing single copy of PPV4 VP and VP2 gene and its immunogenicity studiesVP2 and VP gene were cloned into pFastBac vector by digesting with BamHⅠ and HindⅢ to obtain recombinant transfer plasmids of pFBD-VP2 and pFBD-VP. Which were transformed tinto DHlOBac competent cells, extracted bacmids after the blue-white screening and purification, transfected bacmids into Sf9 cells to obtain recombinant baculovirus rBac-VP2 and rBac-VP. After ultracentrifugation, electron microscopy were performed, results showed that VP2 and VP proteins formed. Emulsified virus-like particles with equivalent freund’s adjuvant and immuned mice, the animal experiment showed that certain level of ELISA antibody anti-PPV4-VP protein was produced, cellular immune response were also induced.3. The construction of recombinant baculovirus expressing three copies of PPV4 VP and VP2 gene and its immunogenicity studiesVP2 and VP were cloned into pFastBac vector separately by digesting with BamhⅠ / EcoRl, SpeⅡ / NotⅠ, SalⅠ / HindⅢ to obtain three copies of recombinant plasmids pFBD-3VP and pFBD-3VP. Which were transformed into DH10Bac competent cells, extracted bacmids after the blue-white screening and purification, transfected bacmids into Sf9 cells to obtain recombinant baculovirusrBac-3 VP2 and rBac-3 VP. Calculated the infected cells and emulsified with equivalent freund’s adjuvant and immuned mice, the animal experiment showed that certain level of ELISA antibody anti-PPV4-VP protein was produced, cellular immune response were also induced.