Expression Of Porcine Reproductive And Respiratory Syndrome Virus-Nsp12 And Its Application In Detection Of Porcine PRRSV Antibodies In Pig Serum

Porcine reproductive and respiratory syndrome(PRRS)is a disease caused by the porcine reproductive and respiratory syndrome virus(PRRSV)that seriously affects the pig industry.PRRSV is a single-stranded,positive-strand RNA virus with a genome length of 15.1 to 15.5 kb and contains 11 Open Reading Frames(ORFs),ORF1 encodes non-structural protein Nsp1-Nsp12,Among them,Nsp12 is a non-structural protein that has been rarely studied,Previous studies have confirmed that Nsp12 is necessary for PRRSV replication and participates in the synthesis of viral RNA,and Nsp12 is highly conserved.By studying the growth and decline of anti-PRRSV Nsp12 antibodies produced in pigs after PRRSV infection,we can further understand the PRRSV infection in pig farms,which is of great significance for the prevention and control of PRRS in pig farms.This study uses the prokaryotic expression system to express the non-structural protein NPR12 of PRRSV.Then,The expressed and purified Nsp12 protein is used to coat the ELISA plate to detect the antibodies against PRRSV in pig serum,and to provide support for evaluating the growth and decline of anti-PRRSV Nsp12 antibodies produced in pigs after PRRSV infection.The specific process of project implementation is briefly described as follows:1.Prokaryotic expression and purification of PRRSV Nsp12Using the extracted PRRSV RNA as a template,the gene encoding PRRSV Nsp12 protein was amplified by RT-PCR technology.The size of the amplified product was about 480 bp;then,the Nsp12 gene sequence was cloned into the prokaryotic expression vector pET-32 a by restriction enzyme digestion and ligation.Then,the recombinant plasmid was transformed into the E.coli expression host strain BL21(DE3).After induction,SDS-PAGE analysis was performed.The size of the expression product was about 40 k Da(including the tag protein of the expression vector and the fusion Protein),repeated exploration to determine the optimal IPTG concentration and culture temperature to induce the expression of Nsp12,and then the expressed Nsp12 was purified by affinity chromatography using a Ni column,and the purified Nsp12 protein was renatured by dialysis.Then,repeatedly explored to determine the optimal IPTG concentration(0.5mmol/L)and culture temperature(37℃)for inducing Nsp12 expression,and then using Ni column to perform affinity chromatography purification on the expressed Nsp12,and the purified Nsp12 protein was renatured by dialysis as a coating antigen for ELISA.2.Detection of swine blue ear disease antibody in pig serumThe purified recombinant Nsp12 protein was used to coat the ELISA plate,and the "checkerboard method" was used to explore the ELISA reaction conditions.92 serum samples(33 clinically confirmed cases)collected from different pig farms were tested,and the results showed that 32 were positive((All are samples of clinically confirmed cases)),60 negative.In this study,the Nsp12 protein of PRRSV was successfully expressed,and the expressed Nsp12 protein can be used to detect antibodies against PRRSV in pig serum,which provides support for further research and evaluation of the growth and decline of anti-PRRSV Nsp12 antibodies in PRRSV-infected pigs.