Isolation And Identification Of Canine Parvovirus And Establishment Of Detect Antibody Method

Canine parvovirus 2(CPV-2)was discovered in the United States in 1982.CPV-2 is one of the major reasons of high morbidity and mortality in dogs and wild animals.In order to investigate the genetic variation of epidemic canine parvovirus(CPV),intestinal tissue and anal swabs of two beagle dogs suspected to died from CPV infection were collected from a beagle dog farm in Jilin Province.The collected samples were filtered and sterilized,and inoculated into F81 cells for isolated.The virus was identified by electron microscopy,PCR,immunofluorescence and animal experiments.The results showed that the F81 cells appear typical CPV cytopathic(CPE)after inoculation.The virus had no capsule with a diameter of 20 nm under electron microscope.PCR results showed that both two strains were positive for CPV and were named as JL-(18)1/Beagle and ZJ-LX respectively.Immunofluorescence assay showed that both two strains of canine parvovirus could react with CPV monoclonal antibody and produce green fluorescence.Animal experiment showed that both JL-(18)1/Beagle and ZJ-LX could infect beagle dogs and generate typical clinical symptoms of canine parvovirus infection.The genetic evolution of VP2 gene of JL-(18)1/Beagle and ZJ-LX was analyzed by DNAstar,the results showed that JL-(18)1/Beagle belonged to New CPV-2b,and ZJ-LX belonged to CPV-2.JL-(18)1/Beagle was closely related to CPV/CN/JL3/2013,CPV/CN/HB1/2013 and CPV/CN/JL6/2013,and the VP2 gene of ZJ-LX shared the same evolutionary branch with raccoon dog parvovirus from Hebei Province.Compared with recent CPV isolates of Jilin Province,the key amino acid sites of JL-(18)1/Beagle had no significant changes with them.Compared with the original CPV-2,four amino acid sites of ZJ-LX,I219 V,N375D,V562 L and Y573 F were mutated.In addition,two new nucleotide mutations T714 C and A1098 G were also found in the NS1 gene of ZJ-LX.The recombinant prokaryotic expression plasmid p ET-30 a was constructed for ZJ-LX strain,and the recombinant p ET-30a-VP2 protein was expressed in large quantities.The VP2 gene of ZJ-LX was amplified by specific primers containing the restriction sites of Eco RI and Hind III.The PCR product of VP2 was connected with p ET-30 a vector after double digests,the recombinant prokaryotic expression plasmid p ET-30a-VP2 was constructed for ZJLX strain,and then transformed into E.coli BL21(DE3).The recombinant proteins were collected after IPTG induction.SDS-PAGE analysis showed that the protein expression of VP2 was the highest when the inducer concentration was 0.5 mmol/L,the induction temperature was 37 ?,and the induction time was 4 h.p ET-30a-VP2 recombinant protein were obtained by optimal conditions and purified,and identified by western blotting.The results showed that the recombinant VP2 protein could react with His and VP2 monoclonal antibody respectively.The results indicated that the p ET-30a-VP2 recombinant protein had good reactionogenicity.In the presenrt study,the purified recombinant VP2 protein was used as the coated antigen to establish an indirect ELISA method for detecting CPV antibodies.The method was optimized by screening the reaction conditions.The method was applied preliminarily,and the specificity,sensitivity,and reproducibility were assessed.The results showed that the optimal antigen coating concentration of CPV antibody indirect ELISA method was 6.5 ?g/m L,the optimal antigen coating time was 12 h,the optimal blocking solution was 5% skimmed milk,the working concentration of the primary antibody was 1:16000,the optimal incubation conditions were 37 ?,1 h,the optimal incubation conditions of the second antibody were 37 ?,1 h.The specificity test results showed that the indirect ELISA method could detect CPV antibody specifically,and there was reaction with the positive serum of CAd V-1 and CDV.The variation coefficients of intra and inter batch repeatability were less than 10%.Monoclonal antibody against VP2 was detected at a minimum concentration of 0.03?g/m L.Compared with neutralization test(SN),the sensitivity was 91.2%,the specificity was 80%,and the coincidence rate was 89.7%.This study laid a foundation for the establishment of CPV antibody detection method and the development of genetic engineering vaccine.