An IgG Antibody Detection Kit Of Canine Parvovirus

This study aims to research a kind of canine parvovirus IgG antibody detection kit,The faeces samples were collected from a dog with severe diarrhoea of a suspected by canine parvovirus(CPV) infection disease. with cat kidney F81 cell isolation and culture, the virus nucleic acid and serological identification F81 cell to F4 generation CPV virus infection after different time collecting cells and culture supernatant, immune peroxidase were used respectively to monolayer cell staining(IPMA) and PCR detection of virus proliferation dynamics, virus respectively with serum free medium and serum culture medium for in vitro culture, determination of IPMA virus drops. Design specific primers, PCR amplification CPV isolates, VP2 gene cloning PCR products to pMD18 T carrier, carrier recombination enzyme digestion and sequence identification sequencing, DNA sequences using DNAStar7.0 and Mega5.0 software similarity and evolutionary analysis results show that the success a CPV virus strain was isolated, named CPV-NY, the strain in F81 cell proliferation can produce significant cytopathic effect(CPE). Can adopt IPMA from 6h after infection. F81 cell virus detection. cells a large increase in the amount of virus in the 24 h, PCR detection results show that 4h no virus detected in the cell culture supernatant, after 6h to detect disease and viral loads increase with the increase of time The strain F3 generations using serum culture medium after 72 h, value-added drops to 106.25 / mL, virus serum free medium culture value drops to 105.36 / mL. The strain of VP2 gene open reading frame(ORF) of 1755 bp, coded 584 aa, evolution analysis shows that the strains belong to CPV2 a type In Nanyang region successfully isolated a strain 2 a type of CPV. This study is aimed to prepare the recombinant VP2 protein(rVP2) encoded by a high pathogenic CPV strain(NY strain) by prokaryotic expression system. A recombinant plasmid pET28a-CPV-VP2 was constructed and transformed into E. coli BL21(DE3), rVP2 was expressed at different temperature, or with different IPTG concentration and with different induction time, SDS –PAGE and Western blot were used to analysis expression and antigenicity of rVP2. The results showed that the molecular weight of the r VP2 was about 72 kDa, the protein existed in the form of inclusion body under different conditions. The expression levels showed the highest with 0.2 mmoL/L final concentration of IPTG, induced for 4 h at 37℃. rVP2 can not only react with His-tag monoclonal antibody but also can react with specificity positive serum of CPV, rVP2 showed good immunoreaction activity, The target protein mixed with adjuvant, The preparation of immunogen by emulsify, The preparation of VP2 protein polyclonal antibody by immune rabbit,, Detection of antibody immune activity, antibody titer and virus titer by IPMA. The results showed that the Prokaryotic expression of protein existed in the form of inclusion body, The molecular weight is about 72 KD; The preparation of polyclonal antibody titer is 1600, the virus titer is 107 TCID50 / mL, The antibodies presents specificity reaction with The in vitro cultured CPV and stable VP2 protein expression cell, CPV VP2 protein polyclonal antibody showed good immune activity and specificity, Then developed an IgG antibody detection kit of canine parvovirus,and the assembly of the kit,the same and defferent batch of repeatability,shelf life and are tested for clinical detection of serum.The experiment proved that there were goog repeatability,long shelf life and high sensitivity of IPMA detection kit, providing foundation for the diagnosis genetic vaccine of CPV.