The Establishment Of ELISA Method Using PRRSV Recombinant NSP7for Detection Anti-PRRSV Antibody

Porcine reproductive and respiratory syndrome (PRRS) continues to be one of the most devastating diseases of swine throughout the world. PRRSV is a small enveloped virus containing a positive-sense, single-stranded RNA genome about15kb in length, which contains nine known open reading frames (ORFs). The NSP7protein was encoded by ORFla which are located on the5’end of the genome. The current study show that NSP7is the most suitable antigen for diagnostic development, with the following characteristics:NSP7is expressed as a soluble recombinant protein in bacterial culture, which is convenient for ELISA antigen preparation, especially when applied to diagnostic tests dealing with massive numbers of diagnostic samples; the PRRSV NSP7protein coding region is more homologous among different strains within the genotype; it is able to detect antibody responses later than126days post-inoculation.The nonstructural protein7gene of PRRSV BJ-4strain was amplified by RT-PCR with a pair of specific primers, and then the product was cloned into pET28a vector and sequenced. The plasmid pET28a-nsp7was transformed into BL21(DE3) for expression under induction of IPTG. The result of SDS-PAGE analysis revealed that the molecular weight of the expressed product was33KDa. The expressed protein was purified in one step using Ni-NAT affinity chromatography. Western blot analyses showed the recombinant protein had good immunoreactivity with the reference positive serum.Using the recombinant NSP7as coated antigen, indirect ELISA method for detecting PRRSV antibody of pig serum was established through optimizing the reaction conditions of each step. The NSP7-based ELISA and N-ELISA kit from IDEXX were parallel tested using420clinical porcine serum. The positive and negative samples coincidence rate is87.59%and76%respectively. The total coincidence rate achieves86.9%, indicating that the NSP7-ELISA has the high specificity and the sensitivity.In this study, The prokaryotic expression plasmid pET28a-nsp7for producing NSP7protein was constructed and the recombinant protein with a molecular weight of33kDa was expressed successfully in E.coli, which was recognized by the porcine positive serum in western blot analysis. We investigated the presence of antibodies against PRRSV of420field serum samples by indirect ELISA (iELISA), which were based on NSP7protein. Extensive validation of iELISA was undertaken, with special emphasis on repeatability, cross-reactivity, and diagnostic accuracy. This assay was validated by comparison with IDEXX HerdChekTM ELISA.