Establishment of an indirect ELISA with the major epitope domain of NS1 of PPV expressed in prokaryotical expression system.

According to the amino acid sequence in GenBank of China strain porcine parvovirus,the major epitope domain in NS1 protein was discovered by the software of the molecular biology.A pair of specific primer was designed to amplify the major epitope domain of NS1.The PCR product of interest was size of 843 base pairs and was cloned into the pET30a(+) vector.The prokaryotic expression plasmid pET30aNS1 was successfully constructed and transformed into E.coli BL21(DE3) pLysS competent cells and induced with IPTG.The size of recombinant protein is 43 000 according to being expected.The parameters including cultivation temperature and concentration of IPTG were optimized,then protein of interest was highly expressed,suggesting that the recombinant protein hold a favourable immunogenicity confirmed by Western-blot.Then an indirect ELISA method was established to detect antibody against PPV with the purified NS1 protein as a coating antigen.The result showed that the optimal concentration of coated antigen was 2 mg/L and the optimal dilution of serum was 1∶100.The positive criterion of the ELISA assay was D450≥0.36 and D450 positive serum/D450 negative serum2.In 60 positive sera samples by the indirect ELISA,there are 45 positive sera by Western-blot.It was conclude that the indirect ELISA established in this experiment is more sensitive than western blot.Compared with commercial kits,the assay showed a correlation rate of 96% based on the data obtained from 100 samples.The results indicated that the ELISA is sensitive and specific,and suitable for routine diagnostics and epidemiological surveys.