Construction And Immunogenicity Study Of Canine Parvovirus-like Particles Harboring Epitopes Of Canine Distemper Virus

At present,CDV/CPV is epidemic in the giant panda,without special vaccine production.In addition,the conventional attenuated vaccine has inadequate immunogenicity and risk of reversion to virulence.Virus like particles（VLPs）have a natural virus particle structure without genetic material,so they have better safety and immunogenicity.Therefore,studying these VLPs is of great guiding significance for the development of CDV/CPV vaccine for giant pandas.In this study,CPV-VLPs from giant pandas were prepared by prokaryotic expression to display the antigen epitopes of CDV.The immunogenicity of VLPs was tested by animal immunity experiments to evaluate its potential as a candidate CDV/CPV vaccine for giant pandas.1.Optimization and purification of CPV-VP2 expression conditionsThe VP2 gene of CPV from giant panda was inserted into pCold Ⅰ expression vector,and the recombinant plasmid pCold Ⅰ-VP2 was constructed.By optimizing the expressing host bacteria,IPTG concentration,co-expression of molecular chaperone and solubility-enhancing tag,the solubility of the target protein was improved.The trigger factor（TF）solubility-enhancing tag was ultimately added based on the recombinant plasmid pCold Ⅰ-VP2,and transformed into BL21（DE3）host bacteria.At15℃,0.2 mm of IPTG was used to induce expression,and a large number of soluble VP2 proteins were obtained.2.Preparation of CPV-VLPs with chimeric CDV epitopesVP2 protein is the main capsid protein of CPV,and VP2 protein expressed in vitro can self-assemble into CPV-VLPs under certain conditions.At the N-terminal and loop2 of CPV-VP2,the neutralization epitope of CDV were inserted successively to form c-VLPs.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the solubility of VP2 protein was almost unaffected by the insertion of CDV antigen epitope.Western blotting was used to analyze the recombinant protein after enzyme digestion and purification.The recombinant protein can combine with CDV/CPV antibody,presenting correct-sized specific bands.Hemagglutination test indicated that the purified recombinant protein had similar hemagglutination activity to natural virus.Under the transmission electron microscopy,the VLPs formed round particles with a diameter of about 25 nm,and the morphology was similar to CPV natural virus particles,indicating that the chimeric VLPs were successfully prepared.3.Immunogenicity of VLPsEnzyme-linked immunosorbent assay was utilized to detect the antibody titer of immuned mice.VLPs in each group can induce high level of CPV antibody,and the immunogenicity was similar to that of attenuated vaccine.The CDV antibody level in the VP2N-VLPs group was slightly higher than that in the VP2L-VLPs group.The changes of hemagglutination inhibition（HI）titer in sera in all immune groups reached the peak value at the 42th day(about 1:2¹¹).The average neutralizing antibody titer in the VP2N-VLPs group was 1:2^6.65,slightly higher than that in the VP2L-VLPs group(1:2^6.58)and attenuated vaccine group(1:2^6.21).Lymphocyte proliferation test verified that spleen lymphocytes of mice immunized with VLPs in two groups could proliferate rapidly after antigen-specific stimulation.In this study,CPV-VLPs from giant panda were successfully constructed.Animal experiments verified that the chimeric CPV-VLPs could induce specific humoral and cellular immune responses,which are potential to be a new subunit vaccine with high efficiency and safety.