| Canine parvovirus(CPV)has been considered to be an important pathogen,which can cause acute infectious disease in canids since its emergence in 1978.Though commercial live vaccines are effective in preventing CPV infection,there still exist some safety problems.Thus,it is significant to develop alternative vaccines,such as virus-like particles(VLPs)based vaccine.It represents a promising vaccine approach with good safety and immunogenicity,but has not been available for CPV prevention.VLPs are composed of viral structural proteins but lack viral genome.They could be recognized easily by the immune system and show dramatic effectiveness.In this study,a subunit vaccine against CPV based on virus-like particles(VLPs)with good safety and immunogenicity is reported:1.The soluble VP2 protein was expressed by co-transformation with pTf16 in Escherichia coli(E.coli)and confirmed through SDS-PAGE and Western blot.The result showed that a 70 kDa protein was produced.The yield of soluble VP2 protein was improved after co-expression with Tf16.2.The VP2 protein with an N-terminal His tag was purified by Ni-NTA affinity chromatography.Under the different assembled condition,VLPs were analyzed by dynamic light scattering(DLS)and transmission electron microscopy(TEM).Western blot analysis confirmed that the protein is the target protein by using an anti-CPV polyclonal antibody.The purity of it could be about 90%.DLS and TEM results indicated that VLPs assembly could be affected by PH and NaCl.The VLPs co-expressed with Tf16 had similar size(25 nm)and shape with the authentic virus capsid at 250 mM NaCl PH 8.0.3.After the vaccination of guinea pigs,anti-CPV antibody titers were detected by neutralization and hemagglutination inhibition(HI)tests.Cytokines from spleen lymphocytes of guinea pigs were measured by ELISA.Immunization with these particles could induce high-titer hemagglutination inhibition(1:12288)and neutralizing antibodies(1:6144)in guinea pigs.Splenic cells of them could secrete IFN-γ and IL-4 after stimulation by CPV.In summary,the CPV VP2 protein co-expressed with Tf16 in E.coli could assemble into VLPs similar with natural CPV virions in size and shape.The VLPs could stimulate strong immune responses in guinea pigs against CPV.Thus,the Tf16 co-expression system provides a new approach for producing CPV VLPs with high yield and efficiency.With the following improvements,a CPV VLP-based vaccine may be developed successfully in the future. |
