Establishment And Primary Application Of Double Antigen Sandwich ELISA For Detection Porcine Reproductive And Respiratory Syndrome Virus Antibody

Porcine reproductive and respiratory syndrome (PRRS) is a serious infectious disease of swine which was caused by porcine reproductive and respiratory syndrome virus (PRRSV) and causing high economic losses in the pig industry in the world. The syndrome is characterized by respiratory disease leading to increased mortality in young pigs, respiratory disease leading to decreased performance in adults, and infertility or late-term abortions in sows. There are many methods for diagnosis of PRRS, for example Enzyme linked immunosorbent assay(ELISA), serum virus neutralization(SVN), indirect fluorescent antibody(IFA), immunoperoxidase monolayer assay(IPMA)and so on, these methods existence many of question on sensitivity, Specificity, the cost and time. Now there was no ideal method existence for diagnosis, Prevention and control on PRRSV, According to the epidemiological study and monitoring can given reasonable method for prevent the infection of PRRSV.Nucleocapsid (N) protein was one of the major structural proteins of virus, encoded by ORF7 which has highly immunogenic and conserved among North American and European virus. According to the published genomic sequence of PRRSV VR-2332 strain, Nucleocapsid-specific primers were designed containing restriction enzyme sites for BamH I (forward) and Sal I (reverse). The total RNA that extracted from PRRSV cell cultures as template, The genomic encoding regions ORF7(N gene) of PRRSV strain were successfully amplified by RT-PCR, Then cloned into the multiple cloning site of pGEM-Teasy vector, and transformed into Escherichia coli Top10 competent cell, and subcloned into expression plasmid pGEX-6p-1.The recombinant plasmid pGEX-6P-N was constructed successfully, and transformed into Escherichia coli BL21(plyss) induced by IPTG at 37℃for expression. The results of SDS-PAGE indicated that the combined genes of orf7 Glutathion-S-transferase (GST) was expressed in E.coli BL21(plyss) as a fusion protein GST-N about 41kDa in molecular weight, At the IPTG concentration of 0.8mol/L and after induced 6h, the maximum protein expression amount arrived. By analysis of indirect ELISA, the recombinant protein has well react with positive serum, it conclude that the recombinant nucleocapsid protein could be used as antigen in diagnostic assays for the detection of antibodies of PRRSV.The recombinant nucleocapsid protein was purified by affinity chromatography. Part of recombinant protein as coating antigen, another part of protein was labeled by HRP,A rapid, simple, double antigen sandwich ELISA for anti-N antibodies was developed using a recombinant PRRSV nucleoprotein.It was shown that the optimal concentration of recombinant N protein for coating of plate was 2.2 mg/L, carbonate buffer (pH 9.6) was the best buffer for coating, the optimal coating concentration of antigen and the dilution of serum was determined by checkerboard titration.4℃coating 24 hour, chosen 1%BSA as blocking buffer, blocked 12 hour. The dilution of serum sample was 1:40, and the react condition of serum was 37℃30min,the substrate for ELISA TMB reacted at room temperature for 10min before terminated with the stopping solution. The blocking test for the reaction between the PRRSV serum and the recombinant N protein was carried out by using the PRRSV antigen. The results showed that PRRS virus could block the reaction completely. The cross reaction experiment showed that there was no cross reaction between the antibodies against Swine fever virus, Porcine pseudorabies, Porcine parvovirus and Porcine circovirus.A total of 260 serum samples were used for validation of this recombinant nucleocapsid protein based double antigen sandwich ELISA. And the result was compared to the DEXX PRRSV Kit, The developed ELISA was correlated well with the IDEXX HerdChek ELISA kit, the specificity and sensitivity of the ELISA is 86.7% and 94.7% respectively, coincidence is 93.5%. In conclusion, the double antigen sandwich ELISA is sample, rapid, and reliable for the detection of antibodies against PRRSV. It was considered to be a powerful tool for the diagnostics, epidemiological surveys and outbreak investigations of PRRSV.