Purification And Immunogenicity Study Of Canine Parvovirus Virus-likeparticles Expressed In Bacterium

Canine parvovirus (CPV) belongs to Parvovirus genus, the virus mainly cause vomiting, bloody diarrhea, white blood cells reduction and other symptoms in dogs. Canine parvovirus disease can occur throughout the year, with acute pathogenesis, short disease course, strong infectious, high mortality etc. With the constant evolution of CPV, there are several subtypes, result in the immune effect of the attenuated vaccine and inactivated vaccine has declined in, and the routine vaccines is expensive, it impact on the promotion and use of the CPV vaccine. In this study, we expressed VP2 protein in the prokaryotic cells and CPV virus-like particles were prepared in vitro。The results also showed that the CPV VLPs can induce high humoral and cellular immune response after vaccination of mice.1.It is important to study gene mutation of the canine parvovirus pandemic strain for disease prevention and control. We screened out of Sichuan, Gansu CPV positive samples in this study, in which the antigen gene VP2 of CPV was amplified and sequenced. According to the key amino acid sites, the subtypes of positive strains were identified and VP2 gene sequence were analyzed, The sequence comparison shows that the nucleotide homology was about 98.8-100%, the amino acid homology was about 99.6-100% between the isolated strains and the reference strains. The main different amino acid sites exist in 267,324,440 sites. The pandemic strain was CPV-2a strains, Phylogenetic analysis showed that all the isolated strains exist in the China branch. This study provided a reference for the preparation of VLPs vaccine.2. The complete VP2 gene of CPV-2a was cloned into a prokaryotic expression vector pSMK, in 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 JM109 cells.The positive bacteria was verified by PCR, restriction enzyme digestion and sequencing.And then, pSMK-VP2 recombinant plasmidwas successfully transformed into the expression bacteria RIL cells.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 appropriate buffer solution, it was proved that VP2 protein can assembly to VLPs about 20-30nm in diameter.It showed that the morphology of VLPs was similar to natural CPV virus by dynamic laser light scattering and electron microscopy.4. The mice were immunized with VLPs or CPV, resepectively. The anti-CPV antibodies were detected with indirect ELISA,.The results show that there is no significant difference between the antibody titer of mice immunized with VLPs and that of mice immunized with CPV. Lymphocyte subsets test results showed that VLPs can effectively stimulate the humoral and cellular immunity. The lymphocyte proliferation assay showed he spleen lymphocytes of mice immuned with CPV rapidly proliferated.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 CPV 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 CPV vaccine.