Development Of N Protein-based Indirect ELISA For Diagnosis Of Porcine Deltacoronavirus Antibody

Porcine deltacoronavirus(PDCoV)is an emerging porcine enteric coronavirus that infects swine of various ages,and the clinical symptoms caused by its infection are characterized by diarrhea,vomiting,dehydration and even death in swine.This globally prevalent pathogen often leads a great economic loss to pig farms,thereby posing a huge threat to the healthy development of the swine industry.Since the outbreak and spread of this virus in USA in 2014,there is no effective vaccine to control it yet.Thus,the etiological and serological detection is particularly important.The nucleocapsid(N)protein is the most conserved and abundant structural protein in PDCoV virions,and can produce extremely high level of antibodies upon the viral infection to the organism,thereby being considered as the preferred target protein for serological diagnosis.In this study,the PDCoV N protein was cloned into pET28 a vector to obtain a recombinant construct named pET28a-PDCoV-r N.Subsequently,the PDCoV N protein was successfully expressed and purified from prokaryotic cells.Therewith,an indirect ELISA method for the PDCoV diagnosis was successfully established using this N protein as the coated antigen.The main research contents are as follows:1.Prokaryotic expression,purification and identification of the PDCoV N proteinThe full-length PDCoV N gene was cloned into pET28 a vector to successfully obtain the prokaryotic expression plasmid of PDCoV N protein.Subsequently,this recombinant plasmid was transformed into BL21(DE3)competent cells.and results showed the PDCoV N protein was arose in soluble in the supernatant of the cleaved E.coli cells,where could gain high purity protein via nickel column affinity chromatography.The immunization of mice with the purified N protein and the confirmation with Western blot demonstrated that this N protein displayed good immunogenicity and reactivity,as indicated by its specific reaction with positive porcine PDCoV serum.Basically,the purified N protein provided a specific coating antigen for the subsequent establishment of indirect ELISA detection.2.Establishment of PDCoV N-based indirect ELISA systemAn indirect ELISA method was established by using the purified N protein as the coating antigen and the Horseradish Peroxidase labeled staphylococcus aureus protein A(HRP-SPA)as the enzyme-conjugated secondary antibody.Furthermore,the optimized ELISA reaction conditions and procedures should be: coating with 0.125 μg/hole N protein at 4℃ for overnight;blocking with 5% non-fat milk solution for 1 h;incubating with the sample serum at 1:600 dilution for 1h;incubating with the HRP-SPA at 1:7500 dilution for30 min;applying the substrate to the chromogenic solution for 15 min.Consequently,the sensitivity of this established ELISA method reached up to 1:2400 as relative to PDCoV standard positive serum samples,while the variation coefficient in repeatability test was less than 10%,moreover,there were no antigenic cross reactions with PEDV,Po RV,PCV2,PPV,PRRSV positive sera.Additionally,the results obtained by this ELISA method had a 100%,95%,and 97.5% coincidence rate with those determined by RT-PCR in the identification of positive,negative and overall compliance,respectively.Thus,in this study,an indirect ELISA method for the specific antibody of N protein detection was established,which provided the strong basis for the serological diagnosis and prevention and control of PDCoV pathogen.