Construction Of Porcine Parvovirus-like Particles Fused With VP22 Peptide And Its Preliminary Research Of Immunogenicity

Porcine parvovirus(PPV)is a major cause of reproductive failure in swine,and vaccination is the main measure for the prevention and control of this disease.Due to its good immunogenicity and high safety,virus-like particles(VLPs)vaccine has been becoming a hot research direction for all kinds of virus vaccine.Porcine parvovirus virus-like particles(PPV-VLPs)are empty capsid particles without PPV DNA,which are self-assembled by PPV VP2 structural protein(hereinafter referred to as VP2)and are analogous to natural virus particles in morphology,and it has very strong bioactivity and immunogenicity.The tegument protein VP22 of herpes simplex virus 1(HSV-1)can has delivery capability and can increase the immunogenicity of fusion antigens by cross-priming.In addition,HSV-1 VP22 protein(hereinafter referred to as VP22)can enhances immune responses of DNA vaccines.This research is divided into three parts.The details of the research are as below:1.Study on CPP VP22 penetrability into biomembranesThe enhanced green fluorescent protein(EGFP)gene was amplified by vector pEGFP-N1 as the template.The VP22 gene fragment was spliced into the 3 '/5' end of EGFP gene by SOE-PCR to obtain VP22-EGFP and EGFP-VP22 fusion gene.The recombinant plasmids pET28a-EGFP-VP22,pET28a-VP22-EGFP and pET28a-EGFP were constructed by cloning the two fusion genes and EGFP gene into pET-28a(+)expression vector.The recombinant plasmid pET28a-EGFP-VP22 and pET28a-EGFP were sequenced and identified.The recombinant plasmids and pET-28a empty vector were then transformed into E.coli BL21(DE3)and were expressed.The results of SDS-PAGE and Western Blot showed that the molecular weight of the expressed protein was about 51 kDa,51 kDa and 47 kDa,and the size was consistent with the expectation.The expression of EGFP-VP22 and VP22-EGFP EGFP was successful.The expressed protein was purified by Ni-NTA affinity chromatography,and the purified protein was added to Vero cells for co-culture.After 10 hours,the cells with VP22-EGFP and EGFP-VP22 protein were observed to have strong green fluorescence,but no green fluorescence was observed in cells treated with EGFP and blank control group,suggesting that VP22 short peptide could be effective in carnying the protein attached to it through the cell membrane into the cell.2.Construction and identification of PPV-VLPs fused with VP22 peptidePreparation of PPV VLPs was done in Bac-to-Bac baculovirus expression vector system.Genome PPV N was used as template to amplify VP2 gene.The VP22 gene fragment was spliced into the 3 '/5' end of VP2 gene by SOE-PCR to obtain VP22-VP2 and VP2-VP22 fusion gene.The recombinant plasmids pFB-VP22-VP2,pFB-VP2-VP22 and pFB-VP2 were obtained by cloning the two fusion genes and VP2 gene into pFastBac TM 1 vector.The recombinant plasmid was transformed into DH10Bac competent cells for homologous recombination to obtain recombined Bacmid-VP22-VP2,Bacmid-VP2-VP22 and Bacmid-VP2;Recombinant Bacmids was transfected into sf9 cells by liposome method to obtain recombinant baculovirus rBac-VP22-VP2,rBac-VP2-VP22 and rBac-VP2.Recombinant baculovirus strains were infected with sf9 for recombinant protein expression.The results showed that VP2-VP22,VP22-VP2 and VP2 protein were successfully expressed in the bands molecular mass(Mr)of about 67kDa,67kDa and 64kDa,which were consistent with the expected.The recombinant baculovirus infected cells were further identified by IFA,and specific fluorescence was observed.The expression of recombinant protein was reconfirmed to be biologically active.Electron microscopy shows that the VP2 and VP22-VP2 protein assembled into spherical particles with diameters ranging from 22-24 nm,which was similar to that of native PPV,however,VP2-VP22 did not assemble the virus-like particles.The result indicates that VP2 N-terminal fused with VP22 did not affect the ability of VP2 to form virions,while C-terminal fused with exogenous proteins may affect VP2 in the formation of VLPs.3.Preliminary research of immunogenicity of PPV-VLPs fused with vp22 peptideThe VLPs-VP22-VP2,VLPs-VP2 and protein VP2-VP22 were simply purified by saturated ammonium sulfate method.Adjuvants Freund',ISA 206 and white oil-Span80 were added to VLPs-VP22-VP2 or VLPs-VP2 or protein VP2-VP22,respectively,then,the formulations were used to vaccinate mice,with the PPV oil inactivated vaccine and PBS as control.Therefore the immunogenicity of the three recombinant proteins was compared and the immunological adjuvants were screened.The PPV specific antibody of the immunized mice was detected by indirect ELISA.The results show that VLPs-VP22-VP2 and VLPs-VP2 can induce the production of specific PPV antibody in mice,and the antibody level corresponding to ISA 206 adjuvant is similar to that of PPV inactivated vaccine,while the recombinant protein VP2-VP22 antibody level is significantly lower than PPV inactivated group.IL-2 is a major growth factor for T cell proliferation.IFN-y can only be produced by only activated T cells and natural killer cells(NK cells).The levels of IL-2 and IFN-y in the collected serum of the immunized mice show that VLPs-VP22-VP2,VLPs-VP2 and VP2-VP22 immunoglobulin can effectively stimulate the production of IL-2 and IFN-?,and the stimulation power of VLPs-VP22-VP2 fused with VP22 in producing IFN-y and IL-2 is significantly higher than that of VLPs-VP2 and PPV inactivated vaccine(P<0.05).In addition,the protein group added by ISA206 adjuvant has superior effect to groups with other adjuvants.In conclusion,the protein fused with VP22 protein did not significantly improve the humoral immunity of VP2 protein,but it could significantly improve the cellular immunity of VP2 protein,and the VP2 N-terminal fusion protein has better effect than C-terminal fusion protein.VLPs-VP2,VLPs-VP22-VP2,when added by ISA 206 adjuvant,can induce the mouse body more effectively to produce PPV-induced humoral and cellular immunity after vaccination.