Isolation And Identification Of Canine Parvovirus YBYJ Strain And Cloning Of The VP2 Gene And Preparation Of Its Polyclonal Antibody

Canine parvovirus is a pathogen, which caused two distinct presentations of canine hemorrhagic enteritis and non-suppurative myocarditis among dogs and carnivores, and is one of the most transmissible diseases in the Parvovirus family. The CPV was first isolated and identified from a dog with diarrhea disease and leucopenia in 1978. To differentiate it from CPV-1, the new virus was named CPV-2. CPV-2 is a non-enveloped, single-stranded DNA virus, as a highly infective virus, causing high incidence and mortality rates, and widely spread in the world among young dogs. In recently years, with the quickly mutant amino acids of CPV-2 capsid protein, new strain of antigenic variants CPV-2a, CPV-2b, CPV-2c, CPV-2c(a), CPV-2c(b) was reported in the research field of CPV. And the new mutant antigenic had gradually replaced traditional CPV-2 become the epidemic strain. It is seriously damage to the canine health, and influence the economic development.In the study, in order to known the locate CPV genetic evolution and developed the new vaccines, the suspected virus was isolated from infected F81 cell, and identified by IFA, Animal regression tested, Hemagglutination (HA) tested, and then cloned and analyzed the complete CPV-VP2 gene for further investigating, and construct the sub-cloned pGEX-4T-1 prokaryotic expression vector(pGEX-CPV-VP2), the target protein was induced expressing in E.coli BL21, and the recombinant expression protein was purified and identified by SDS-PAGE, then preparation of polyclonal antibody by immune mice, measured the result by ELISA. The result showed that, successfully isolated and identified the strain of YBYJ CPV;and the CPV-VP2 gene was amplified with the length 1755 bp, contains 584 amino acids; and the homologous analysis of CPV-VP2 nuclear and amino acids sequences was 98.7～99.9%,96.9～99.8% respectively within samples and references strains; and showed that the mutation of CPV-VP2 a little from the analysis result among different samples; phylogenies analyses of the CPV-VP2 indicated that the strain of YBYJ related to CPV-2a branch, and not obviously with the other branches, share the nearest relationship with China isolation samples like Beijing(EF666063)、 Xinjiang(EU170352)、Changchun(GU380301)、Guangzhou(KF785793) and Shanxi(J N403045); the recombinant plasmid pGEX-CPV-VP2 was constructed successfully, the recombinant protein with a relative molecular weight of 118 Ku was identified by SDS-PAGE and Western-blot analysis indicated that this fusion protein was specifically reached with parvovirus positive serum, investigated that the recombinant protein shown good reactionogenicity and biological activity, which could be used to identified as antigen in the diagnostic of CPV.All the work will laid foundation for further research and the study of recombinant vaccine and new diagnose reagent. And it will be benefit for canine breeding of the Yanbian region.