Prokaryotic Expressions And Establishments Of Indirect ELISA Of NS1and VP2Gene Of Porcine Parvovirus

This study was to monitor the PPV antibody levels and its prevalence by establishing two indirect ELISA with recombinate PPV NS1and VP2proteins as antigens detecting swine anti-NS1protein and anti-VP2antibodies.This study firstly detected the diseased materials that our laboratory saved by PCR, and screened the PPV-positive ones; secondly, we compared the homology of the PPV sequences which landed in the GenBank by software and predicted the major antigenic regions of the NS1and VP2proteins; thirdly, we amplified them and cloned them into the vector pET28a (+) and constructed two recombinant plasmids associated with the NS1, and they were the pET28-NS1①plasmid containing its ORF1-1218bp fragment and the pET28-NS1②containing its ORF1294-1983bp fragment. At the same time, we constructed three recombinant plasmids associated with the VP2, and they were the pET28-VP2①containing its ORF full-length fragment and pET28-VP2②containing its ORF451-1737bp fragment and pET28-VP2③containing its ORF781-1737bp fragment.These five recombinant plasmids have been expressed in E. coli bacterias of BL21(DE3) and identified by SDS-PAGE electrophoresis. But the results were that only pET28-NS1①, pET28-VP2②, pET28-VP2③can express, and the molecular size were52kD,40kD,50kD respectively, and all of them were inclusion bodies.By Western Blot analysis, it showed that the immunogenicities of all these three recombinant proteins were excellent.And we purificated them using Ni2+affinity chromatography.According to the methods that introduced by "The ELISA Guidebook ", we established the PPV NS1and VP2indirect ELISA methods, and explored and optimized conditions of the test. Finally, we finalized the conditions of NS1-ELISA were that the antigen was200ng/hole, the serum dilution factor was1/50, the HRP secondary antibody dilution was1/750, serum reaction time and the HRP secondary antibody time were45min, the reaction time of TMB was10min. The conditions of PPV VP2indirect ELISA were that antigen was400ng/hole, the serum dilution factor was1/100, the HRP secondary antibody dilution factor was1/750, the serum reaction time and the HRP reaction time were30min, the TMB reaction time was10min.Through the experiments that checking the NS1-ELISA and VP2-ELISA tests, the results showed that the sensitivity and specificity of these two proteins were fine, and the efficient variations were less than10%, indicating that the repeatability were good. The results compared with the one detected by the kit of Switzerland Prio CHECK PPV showed that the consistency of these two ELISA we established were high in the latter of pigs'growing period, but there was a big difference in the early stage of maternal antibodies. The reason maybe was that the recombinant proteins expressed as forms of inclusion bodies which were linear epitopes, but the maternal antibodies of PPV played a role with structure epitopes mainly. But it also provided a reference for expressing NS1and VP2protein of PPV and establishing ELISA methods, and it pointed out the direction that to build the next step of refolding protein and promoting soluble expressions.