The Influence Of Formation Of PPV VPLs By Inserting PCV2 ORF2 Into PPV Strain SC-1 VP2 At Different Site

In this experiment, the amplified VP2(PPV strain SC-1) gene and ORF2 gene (PCV2 strain SC) were clonged into pMD18-T Simple vector respectivly, then VP2 gene was inserted by ORF2 gene at Hindâ…¢and SacI position respectively (located at 1/3 N terminus and 1/3 C terminus of PPV VPLs) and the recombinant plasmid pPVP2-ORF2.H and pPVP2 -ORF2.S were obtained. Transfer the recombinant genes into eukaryotic expression vector pEGFP-C1 to construct the recombinant plasmids pEGFP.VO.H and pEGFP.VO.S. Then the eukaryotic expression recombinant plasmids were transfected into Cos7 cells with LipofectamineTM2000 and the recombinant genes were expressed and detected by fluorescence inverted microscope directly. Cells which showed obvious green fluorescent were prepared for electronmicroscope observation. Healthy mice were immunized with pEGFP.VO.H and pEGFP.VO.S DNA, also with pEGFP-VP2 DNA,pEGFP-ORF2 DNA,pEGFP-C1 DNA, Porcine Parvovirus vaccine,attenuated Porcine Circovirus and 0.9% NaC1 as control.The dynamic variation of blood T lymphocytes and the antibody variation of PPV and PCV2 in serum were measured.The results showed that: the eukaryotic expression recombinant plasmids pEGFP.VO.H and pEGFP.VO.S were constructed successfully and the fusion expression of green fluorescent protein was observed. VLPs were observed in sample pEGFP.VO.H only. as for T lymphocyte of peripheral blood of immunized mice, the ratio of CD₃⁺, CD₄⁺ T lymphocyte of group pEGFP.VO.H araised in a certain degree but CD₈⁺T lymphocyte fell 7-14d after immunization and then raised.When turned to group pEGFP.VO.S, the ratio of CD₃⁺ and CD₄⁺ was similar to group pEGFP.VO.H's ,but CD₈⁺T lymphocyte almost descend.At the same time,relatively high level of PPV, PCV2 specific antibody was detected from serum of group pEGFP.VO.H and pEGFP.VO.S. It was showed that inserting ORF2 polypeptide into PPV VPLs at the site of 1/3 of N termini could make parvovirus-like particles assemble spontaneously and specific antibody response of PPV and PCV2 were induced also.lt indicat that the 1/3 N terminus in PPV empty capsids is an adequate site for insertion of PCV2 ORF2 polypeptide.On the contrary, the site of 1/3 C termini of PPV VPLs could not be used for epitope fusion.