Preparation Of PPV VP2 Double Antibody Sandwich ELISA Diagnostic Kit

Porcine Parvovirus infection caused reproductive disorders which seriously restricting the development of Chinese pig industry, various strains and different ages of pigs could be infected with PPV, the main source of infection is infected pigs, therefore, established the detection method of quickly and accurately diagnose PPV infection is especially important. Diagnosis of porcine parvovirus most commonly used detection methods include hemagglutination and hemagglutination inhibition test, neutralization test, virus isolation test, latex agglutination test, immunofluorescence test and detect nucleic acid probe technology, but these methods or time-consuming, or require special equipment and professional technically operation, or a lower detection rate, or the high cost. This study used PCR amplification PPV VP2 full-length, amplification product was cloned into the expression vector pET-32 a and transformed into E.coli.BL21. Different induction times, IPTG concentration and temperature induced respectively VP2 fusion protein, by SDS-PAGE electrophoresis gel extraction purified VP2 fusion protein and immunized rabbits, prepared anti-PPV VP2 polyclonal antibody. Screened PPV VP2 double-antibody sandwich ELISA diagnostic kit for the best working conditions, and examined its specificity, repeatability, stability, sensitivity, and consistent rate. Trying to develop a simple, rapid, high detection rate and lower cost of PPV VP2 double-antibody sandwich ELISA diagnostic kit, lay the foundation for rapid and accurate diagnosis PPV infection. Research has made the following results.1. PCR amplified 928 bp and 873 bp DNA fragment of the anterior and posterior segment, fusion PCR spliced to obtain the full-length of 1740 bp VP2; connected the VP2 and prokaryotic expression vector pET-32 a, successful constructed the recombinant plasmid, named pET-32a-VP2. VP2 fusion protein optimized the best expression conditions: 37 °C, concentration of IPTG induction of 1.0 mmol/L, induced time for 4 h. Cut from SDS-PAGE gel to purified VP2 fusion protein.2. Purified VP2 fusion protein inoculated the rabbits, indirect ELISA detection of anti-PPV VP2 polyclonal antibody titer of 1:12 800; polyclonal antibodies were diluted 1:1 000 can specifically bind with PPV of PK-15 cells total protein; polyclonal antibodies were diluted 1: 250 can specifically bind with PPV, and did not specifically bind with porcine circovirus type 2, porcine reproductive and respiratory syndrome virus, transmissible gastroenteritis virus.3. The best working conditions for double-antibody sandwich ELISA : rabbit anti-PPV VP2 polyclonal antibody the best dilution of 1: 3 200, monoclonal antibody the best dilution of 1: 6 400; HRP-goat anti-mouse IgG labeled as the best dilution of 1: 8 000; HRP best time was one hour; the best blocking buffer was 5% skim milk, the best closure time was one hour; TMB the best reaction time was fifteen min. Detected the establishment of the PPV sandwich ELISA diagnostic kit specificity, repeatability, stability, sensitivity, and coincidence rate. Compared with commercially PPV ELISA kit, both only reacted with PPV and not reacted with PCV2, PRRSV and TGEV; repeatability test showed that this diagnostic kit OD450 nm value of the coefficient of variation ranged from 1.17%~3.24%, commercially PPV ELISA kits OD450 nm value of the coefficient of variation ranged from 1.04%~3.20%, both less than 5.0% less than 5.0; stability tests showed that the diagnostic kit and commercially PPV ELISA kits continuously measured at 37 °C incubator 7 d, both OD450 nm values were no obvious change; sensitivity analysis showed that this diagnostic kit and commercially PPV ELISA kits are the minimum detectable amount of 102 TCID50; compared with commercially PPV ELISA kits, this diagnostic kit the positive rate was 92.3%, the negative rate was 97.6%.This study was prepared kit could react with the PPV virus solution, with PCV2, PRRSV and TGEV virus solution was not cross-reactivity. the coefficient of variation of OD450 nm ranged from 1.17%-3.24%; 37 ° C incubator continuous 7 d OD450 nm did not obvious change; the minimum detectable amount was 102 TCID50. The positive rate was 92.3%, the negative rate was 97.6%. The results will provide a fast, convenient, safe and low-cost PPV diagnostic kits for large-scale pig industry in China.