Preparation And Immunogenicity Study Of Porcine Parvovirus Virus-like Particles

Porcine parvovirus(PPV),a widespread infections virus,is one of the causative agents of reproductive failure in pregnant sows.It was characterized by stillbirths,mummified fetuses,and early embryonic death.Classical inactivated vaccine is extensively used to control PPV infection,but problems concerning safety,such as incomplete inactivation may occur.So it is necessary for us to develop a safe,efficient PPV vaccine.Virus-like particles(VLPs)are multiprotein structures that mimic the organization and conformation of authentic native viruses but lack the viral genome.Given their intrinsic immunogenic properties and high safety profile,VLPs have been generated and have been tested as vaccine candidates for a variety of human and animal viral diseases.In this study,VLPs of PPV were generated in the prokaryotic system through expression of PPV VP2 protein.Moreover,the immunogenicity of these PPV VLPs can induce high humoral immune response after vaccination of guinea pig.Which may pave a new way for research and development of PPV VLPs vaccine.1.The VP2 gene of PPV strains was optimized and synthesized according to the E.coli coden usage preference,which were inserted into a prokaryotic expression vector pSMK,which contains with a poly-histamines(6×His)and small ubiquitin-like protein(SUMO)tag.The recombinant plasmid was successfully transformed into the cloning bacteria Trans5a cells.The positive plasmid was designated as pSMK-VP2,which was transformed and expressed in RIL(DE3)competent cells.The protein was identified by SDS-PAGE,results show that there was a more significant band at about 86 ku conforming to the expected size,which indicated that the gene could be expressed in E.coli.2.The induction conditions,including host bacteria?IPTG concentration?bacterial biomass brfore induction?induction temperature?induction time were optimized by sigle-factor design.It is found that the optimal expression condition for the recombinant protein were determined as follows:RIL(DE3)as host bacteria,when the OD600nm reached 0.7,the bacteria liquid was initiated with 0.6 mM IPTG at 16 ? for 18 h.3.Removed the SUMO and His tag in N-terminal of the fusion protein by the SUMO protease,we obtained the VP2 protein without any tag.In pH 7.0?300 mM NaCl buffer solution,it was proved that VP2 protein can assembly to VLPs about 18-26nm in diameter.It showed that the morphology of VLPs was similar to natural PPV virus by dynamic laser light scattering and electron microscopy.The guinea pigs were immunized with PPV VLPs antigen,whereas the PPV inactived vaccine was used as the control antigen.The results show that there is no significant difference between the antibody titer of guinea pig immunized with VLPs and that of immunized with PPV inactived vaccine.In summary,we successfully constructed the prokaryotic expression vector encoded canine parvovirus VP2 gene.VP2 protein expressed and purified can assembly to VLPs in vitro with similar shape as natural PPV virus.VLPs effectively stimulated the humoral and cellular immunie response in mice.This study provided a new idea and reference for the development and application of new type of PPV vaccine.