Preparation Of Canine Parvovirus-like Particles Using Baculovirus Expression System

Canine parvovirus（CPV）is derived from the Feline Panleukopenia Virus（FPV）,the gene sequence similarity between canine parvovirus and feline parvovirus is as high as 99%,but they have different antigenic properties,host range and hemagglutination ability.Since its discovery in 1978,canine parvovirus has spread rapidly around the world and has seriously threatened the development of the kennel Industry.High morbidity and mortality in infected animals are caused by the high rate of virus mutation and the lack of timely vaccination of dogs.At present,the CPV strains circulating in our country are CPV-2a,2b and 2c,while the current vaccine strain is the original CPV-2,whose immunological function is far different from the circulating strains.Therefore,it is urgent to develop a safe preventive vaccine against the infection of epidemic strains.Virus-like particle（VLP）is a protein complex composed of single or multiple structural proteins of the virus.Its morphological structure is similar to that of natural virus particles,but the capsid does not contain the viral genome.In this thesis,self-assembled canine parvovirus VLPs were obtained by insect-baculovirus expression system.The research contents and results contain three main parts:1.Expression of parvovirus VP2 proteinsThe gene sequences for expressing of VP2 protein of CPV-2a/GS-1 strain,codon-optimized VP2 protein VP2.opt and FPV were designed and synthesized.The vectors pQB3a-CPV VP2,pQB3a-CPV VP2.opt,pQB3a-FPV VP2 were constructed with p6.9 and polh tandem promoters to regulate gene expression,and pTriEx-CPV VP2 were generated with T7and p10 promoters to regulate gene expression.The results of agarose gel electrophoresis showed that the VP2 recombinant expression vectors were successfully constructed.IPTG induced the expression of CPV VP2,and found that the solubility of VP2 protein was poor.Besides,it seemed that the His tag could not be exposed during purification,and it was difficult to obtain pure VP2 protein.The pQB plasmids were respectively co-transfected with linearized bacmid into Sf9 cells to obtain recombinant baculoviruses to express CPV VP2 and FPV VP2.Virus titers were determined using endpoint dilution assays.Sf9 cells and High Five cells were infected at MOI=3.The expression product was analyzed by SDS-PAGE,and it was found that the VP2protein was about 64 kDa in size,consisting with the pricdicted molecular weight.The expression level of VP2 protein in High Five cells was significantly higher than that in Sf9cells.At the same time,it was found that after the vAc-CPV VP2 and vAc-FPV VP2recombinant viruses infected cells,the expression of VP2 was higher than that of the recombinant viruses vAc-CPV VP2.opt and vAc-CPV VP2.The results from cells infected with different amounts of viruses showed that vAc-CPV VP2 and vAc-FPV VP2 had the highest protein expression when they were infected at MOI=3 in High Five cells.These results demonstrated that VP2 protein was highly productive under the control of tandem promoters p6.9 and polh,and the expression level of VP2 protein without codon optimization is higher than that of optimized one.2.Purification and detection of CPV VLPThe VLP formed by CPV VP2 were purified by sucrose density gradient centrifugation,and the assembly of VLP was observed by transmission electron microscopy.The results showed that the VP2 protein could be trapped in a 35%-50%sucrose gradient,and the purified VP2 protein was about 90%pure.Transmission electron microscopy showed that the VP2protein could self-assemble into a VLP structure similar to natural CPV in shape and size.These results illustrated that self-assembled parvovirus VLP could be assembled and purified using insect baculovirus expression system.3.Examination of the hemagglutination and immunogenicity of VLPHemagglutination assays detected an average hemagglutination dilution of 1:2⁷ for CPV VLP.Raccoon dogs were immunized twice with the purified VLP.The results showed that the primary immunization induced specific antibodies,and the antibody level detected by the hemagglutination inhibition test after the secondary immunization reached a maximum of1:2¹².The data suggests that the CPV VLP prepared in this study has good immunogenicity.In conclusion,insect-baculovirus expression system was used to express high-level soluble CPV VP2 proteins,which can spontaneously form virus-like particles similar to canine parvovirus virions in size and shape.The raccoon dog immunization experiments showed that the VLP formed by VP2 proteins had good immunogenicity.This study laid the foundation for further development of VLP vaccines for CPV.