The Site-directed Mutation Of Cys-402,214 Of Porcine Parvovirus Strain SC-1 And Preliminary Research On The Effect Of PPV VLPs

Based on the porcine parvovirus(PPV) VP2 gene, site-directed mutation was used for the functional research of Cys on the VLPs higher structure formation and its immunogenicity, to find the suitable exogenous gene insertion site, providing rationale for the multivalent vaccine and antigen transport system which were based on the PPV VLPs. Methods: refered to the PPV VP2 gene sequence registered in GenBank(Genbank register number: D00623), mutation and cloning primers were designed and synthesized. Using overlap extension PCR, cysteine on sites 402 (A) and 214 (B) of PPV VP2 protein were changed into arginine respectively. Then the mutants VP2(A) and VP2(B) were inserted into the eukaryotic expression vector pPI-2.EGFP to construct the recombinant plasmids pPI-2.EGFP.VP2(A) and pPI-2.EGFP.VP2(B), which were then transfected into COS-7 cell, effects of mutation on the formation of PPV VLPs was studied. Balb/c mice were immunized using combinant plasmids; dynamic changes of peripheral blood T lymphocytes and the serum PPV antibody level were monitored, so as to study the effects of mutation on the immunogenicity of PPV VLPs.Electron microscope was used to observe the expression products. VLPs were found in plasmid pPI-2.EGFP. VP2(A) transfected cells, which proved that the Cys-214 was vital for the formation of VLPs, while the mutation of Cys-402 had no effect on the package of VLPs; monitoring of peripheral blood T lymphocytes and the serum PPV antibody level showed that the immune efficiency of pPI-2.EGFP.VP2(B) was obviously lower than that of pPI-2.EGFP.VP2(A), relatively high level of PPV specific antibody was detected only in mice immunized pPI-2.EGFP.VP2(A), indicating that the mutation of Cys-402 had no effect on the correct package of VLPs and its immunogenicity. As a result, this site was a potential exogenous gene insertion site, which was worthy of further study. Integrity of VLPs was the key point of its immunogenicity was further confirmed.