Prokaryotic Expression Nucleocapsid Protein And Establishment Of Indirect-blocking ELISA Of Porcine Reproductive And Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome (PRRS), caused by porcine reproductive and respiratory syndrome virus (PRRSV), was characterized by respiatory disease leading to increased mortality in young pigs, respiratory disorder leading to decreased performance in adults, and infertility or late-term abortions in sows. PRRS was first resported in North America in 1987, and now is widely spread worldwide and has a catastrophic economic impact on the global pig industry. Internationally, several kinds of serologic tests have been established, including enzyme-linked immunosorbent assay (ELISA), serum virus neutralization(SVN), indirect fluorescent antibody (IFA) and immunoperoxidasemonolayer assay(IPMA). Additional tests are available including immunohistochemistry staining (IHC), fluorescent antibody assay (FA), polymerase chain reaction (PCR), virus isolation (VI) and gold colloid technique.According to the published genomic sequence of PRRSV VR2332 strain, a pair of primers was designed and the nucleocapsid (N) protein gene of PRRSV VR2332 strain was amplified by RT-PCR and cloned into the multiple cloning site of pET30a(+)vector. The recombinant plasmid was identified. The recombinant plasmid rpET30a-PRRSV/N was transformed into Escherichia coli BL21(DE3) pLysS competent cell and induced with IPTG. The examination analysis using 12% SDS-PAGE and Western blotting showed that the protein was 19 kDa in size and specifically reacted with anti-PRRSV/N protein monoclonal antibody, indicating the recombinant protein had high specificity of immunereaction and could be used as an antigen for detection of antibodies against PRRSV.A rapid, inexpensive indirect-blocking enzyme-linked immunosorbent assay (IB-ELISA) for detection of anti-N antibodies was developed using the recombinant N protein as the coating antigen. A total of 256 serum samples collected from swine herds in Harbin, Daqing areas were detected for the presence of anti-N antibodies. Of the 256 serum samples, 66 were positive by the IB-ELISA. Of 66 IB-ELISA positives, 63 and 58 were confirmed positive by the IDEXX PRRSV antibody test kit and Western blotting assay, respectively. Therefore, the agreement between IB-ELISA and IDEXX kit and between IB-ELISA and Western blotting assay was 95.5% and 87.9%, respectively. In addition, these 256 serum samples were detected for the presence of antibodies against PRRSV GP5 antigen using the indirect ELISA(48/256) with the partial GP5 protein as the coating antigen. Of 256 serum samples, 48 were positive. To confirm the positive samples, they were tested by IDEXX kit and Western blotting assay. Of 48 ELISA positives, 46 and 34 were confirmed positive by IDEXX kit and Western blotting assay with the agreement of 95.8% and 70.8%, respectively. The data indicated that IB-ELISA was more sensitive and specifical than IDEXX PRRSV antibody test kit and Western blotting assay. It was considered to be a powerful tool for routine diagnostics and epidemiological study of PRRSV infection.