Expression And Immunogenicity Test Of Canine Parvovirus VP2 Gene Based On Baculovirus Expression System

Canine Parvovirus disease is a highly contagious disease of dogs caused by Canine Parvovirus(CPV).At present,canine parvovirus disease is widespread in China,which causes serious economic losses to kennel and pet industry in our country.Vaccine immunization is key to control the prevalence of CPV.Therefore,development of a safe and efficient CPV vaccine is of great significance for the prevention of canine parvovirus disease.In this study,we first obtained the CPV major antigen VP2 gene by PCR amplification.The CPV VP2 gene was cloned into baculovirus multi-gene expression system established by our laboratory and it was identified by digestion and sequencing analysis.Meanwhile a series of recombinant baculoviruses were constructed to optimize the expression of VP2 protein,as BV-p YBDM-IM-polh-VP2,BV-p YBDM-IM-p10-VP2 and BV-p YBDM-IM-polh-VP2-p10-VP2.Sf9 insect cells infected with recombinant viruses can be observed with fluorescence microscope.Genomic DNA was extracted from the supernatant of virus-infected cells and PCR was performed using VP2-specific primers.The results showed gene fragment of about 1750 bp was amplified successfully.It indicates that CPV VP2 gene has been successfully recombined into baculovirus genome.Sf9 cells were infected at MOI = 5,the titers of recombinant viruses were detected with endpoint dilution method and q RT-PCR.SDS-PAGE electrophoresis and Western blotting showed that the target protein of about 65 k Da was successfully expressed,which was consistent with the size of CPV VP2 protein.Thus we successfully expressed the CPV VP2 protein in baculovirus expression system.Meanwhile,the expression levels of VP2 protein in different recombinant baculoviruses were measured by quantitative ELISA.The results showed that VP2 protein concentration of BV-p YBDM-IM-p10-VP2 was 1.292 times higher than that of BV-p YBDM-IM-polh-VP2,The VP2 protein concentration of BV-p YBDM-p10-VP2-polh-VP2 was 1.336 times higher than that of BV-IM-polh-VP2,which indicated that we successfully increased CPV VP2 protein expression level using promoter replacement and double copy gene expression.The purified VP2 protein was mixed with an adjuvant to prepare canine parvovirus genetic engineering vaccine in order to study the immunogenicity of CPV VP2 protein.Mice were immunized by intramuscular injection and booster immunization was taken two weeks after first immunization.The result showed that canine parvovirus-specific antibodies were detected in the serum of immunized mice.After booster immunization,antibody titers of immunized mice were increased significantly,in which the highest antibody titer reaching 1.75.The data showed that CPV VP2 protein has good immunogenicity and can produce high level of canine parvovirus-specific antibodies,which has the good prospect to develop a safe and efficient vaccine for canine parvovirus.