Expression Of PPV Non-Structural Protein (NS-1) And Development Of An ELISA For Differential Diagnoses Of PPV Infection And Vaccination

Porcine Parvovirus (PPV) infection was of economic concern to pig breeder worldwide. It was a major cause of stillbirth, mummification, embryonic death, infertility syndrome (SMEDI). Some types of inactivated PPV vaccines were available. Vaccinated with inactivated PPV was generally considered to offer good protection against PPV infection and was used to control reproductive failure due to PPV. In farms where vaccination was carried out, the majority of vaccinated pigs would be seropositive. Therefor, the virus detection and differentiating serological method which chould distinguish vaccinated from naturally infected pigs were necessary. Non-structural polyprotein (NS) plays the major role in DNA replication, RNA transcription and packaging of the virus. Sinece the inactivated PPV vaccine consisting of semi-purified, chemically inactivated virus chould elicit only antibodies against the structural proteins, assays which specifically detect antibodies against the NS-1 protein may be useful in differentiating vaccinated pigs from infected ones.According to the amino acid sequence of PPV china strain in GenBank, we identified the major epitope domain in NS-1. A pair of specific primer was designed to amplify the major epitope domain of NS-1. The PCR product of 843 bp was introduced into pMD18-T vector. The recombinant clone, pMD-T/NS-1s, was sequenced. The nucleotide sequence of NS-1s was compared and analyzed with that published previously in GenBank, and pET30a/ NS-1s recombinant plasmid was constructed by inserting NS-1s gene into pET-30a vector. The recombinant plasmid was transformed into E.coli BL21(DE3), and was induced with IPTG to express recombinant NS-1s protein. The expression of NS-1 protein was detected by SDS-PAGE and Western blot assay. To characterize the antigenicity of the NS-1s protein, an indirect ELISA (I-ELISA) was developed by coating the ELISA plate with the purified NS-1 protein and used to test 256 filed serum samples and the positive samples were confirmed with the Western blot. The blocking ELISA (B-ELISA) using whole PPV was used as the comparative assay and the haemagglutination inhibition (HI) assay was used as the standard method. The result showed that 843 bp length NS-1s gene from the PPV strain was successfully amplified and cloned. Sequencing result showed 98.6% to 100 % nucleotide sequence identity and 98.5% to 100 % amino acid identity with other Chinese strains. SDS-PAGE result showed that the recombinant NS-1s was expressed successfully in E.coli BL21(DE3), and the high antigenicity were comfirmed by Western blot. According to I-ELISA results, 131 serum samples were tested positive from PPV-infected pigs. The agreement with the Western blot was 87.78%. In the B-ELISA using the whole PPV, 204 serum samples were positive which were 100% agreement with the HI test. According to I-ELISA and B-ELISA, 73 serum samples were from PPV-vaccinate pigs and 131 from PPV-infected pigs.In summary, it chould be concluded the expressed recombinant NS-1s protein had high immunogenicity, and the I-ELISA was sensitive and specific, and suitable for routine diagnostics and epidemiological survey.