Prokaryotic Expression And The Establishment Of An Indirect ELISA Assay For VP2 And NS1 Gene Of Porcine Parvovirus S-1

In this study , the open reading frame of PPV VP2 and NS1 were amplified from the infected PPV ST cells by PCR method .Then the products were cloned in prokaryotic vector and pET32c/VP2 and pET32c/NS1expressed were construed respectively. And the sequence were determined.The recombinant plasmid pET32c-VP2 was transformed into BL21 competent cells and expressed in very high level as inclusion body when induced with IPTG..Western blot analysis proved the renaturation protein has good immunoreactivity against PPV antibody. The indirect ELISA method for the detection of PPV specific antibody in procine serum was established ,after the optional working circumstances for the ELISA assay ( antigen concentration:3.1μg/ml;optimal serum dilution:1:100) with chessboard titration.The positive criterion of this ELISA method is OD待检血清≥0.4且OD待检血清/OD标准阴性值≥2.1. The recombinant plasmid pET32c-NS1was transformed into BL21 competent cells and expressed in very high level by using low-temperature induction to reduce formation of inclusion body. Western blot analysis proved the renaturation protein has good immunoreactivity against PPV antibody. The indirect ELISA method for the detection of PPV specific antibody in procine serum was established ,after the optional working circumstances for the ELISA assay (antigen concentration : 2.5μg/ml ; optimal serum dilution :1:200) with chessboard titration. The recombinant NS1 can be applied in differential diagnosis of PPV infections.It show that detection of antibodies agaist porcine parvovirus nonstructural protein NS1 may distinguish between vaccinated and infected pigs.