Establishment And Preliminary Application Of Indirect ELISA Method Baesd On ORF3 For Porcine Circovirus 2(PCV2)

Porcine circovirus type 2 is a primary causative factor of an emerging swine disease postweaning multisystemic wasting syndrome(PMWS). PCV2 can cause immune suppression, mixed infection and secondary infection consequently seriously harm to the swine industry worldwide. It is particularly urgent to reveal the pathogenesis of PCV2 infection.In our study, a pair of primers(ORF3F and ORF3R) was designed according to the ORF3 gene sequence and EcoR I and Xho I restriction enzyme site was added at the end of the primers. Cell culture-adapted PCV2 strain genomes were used as templates for ORF3 gene amplification. the ORF3 gene was cloned into the plasmid pGEX-6p-1 named pGEX-6p-ORF3. The recombinant plasmids were constructed and confirmed by sequencing. The fusion protein GST- ORF3 was expressed in BL21 cells and confirmed by SDS-PAGE and Western blot.The recombinant ORF3 Protein was used as the envelope antigen, and established the indirect ELISA method. We used the method to detect 80 serum samples, and contrast with the commercial kit. The total masculine ratio was 60%.The compliance rate between two methods was 93%.