Development And Evaluation Of An Indirect ELISA For Detection Of Antibodies Against Porcine Deltacoronavirus Using Recombinant Nucleoprotein Antigen

Porcine deltacoronavirus(PDCoV)is a porcine enteric disease virus which has been proven to be associated with severe diarrhea,vomiting,and dehydration in piglet.PDCoV was initially reported in pigs in Hong Kong in 2012.Since first reported in swine in U.S.A.in February 2014,PDCoV rapidly spread to other states in the U.S.A.It was reported that PDCoV was identified in Jiangsu,Anhui,Jiangxi,Hubei and Haerbin provinces which has became a novel pathogen that causes serious impact on swines in China.It is necessary to develope a simple and rapid serological diagnostic tool for investigation of PDCoV epidemiology.PDCoV N protein which exhibited a high degree of conservation suitable as a diagnostic antigen for detection antibodies against PDCoV.In order to obtain PDCoV N protein,the nucleotide sequence of the entire N gene of PDCoV which was obtained from the GenBank was synthesized based on the codon usage bias of Escherichia coli.The synthesized PDCoV N gene was cloned into the prokaryotic expression vectors pET-32a(with a His-tag)and p-GEX-6p-1(with a GST-tag),respectively.Recombinant plasmids were transformed into E.coli BL21(DE3)cells,and than,N gene expressioin was induced using IPTG.The results indicated that the recombinant N proteins of PDCoV were successfully expressed and both of them were soluble proteins.The recombinant proteins was verified using hyper-immune serum of PDCoV by western blot,which means it can be used as a diagnostic antigen to detect PDCoV antibody.In order to provided a serological diagnostic tool for PDCoV,an indirect ELISA,rPDCoV-N-ELISA,which used rocombinant protein pET-32a-PDCoV-N that was expressed from E.coli,was developed to detect antibodies against PDCoV in this study.rPDCoV-N-ELISA was assessed by the test of response conditions,determination of the cut-off value,specificity test and repetition test.Contrast test used the results of the western blot which was verified by using rocombinant protein pGEX-6P-1-PDCoV-N as the gold standard.The results indicated that the best concentration of antigen for coat is 1 ?g/mL,best concentration of sample is 1/100,best concentration of anti-pig IgG peroxidase conjugate was 1/7 500,best rcaction time of samples and anti-pig IgG peroxidase conjugate was 1 h.Sensitivity and specificity of rPDCoV-N-ELISA relative to western blot was respectively 100% and 90.4% when the optimized PR cut-off value was 48.9.The recombinant protein antigen pET-32a-PDCoV-N showed no cross-reaction with antisera against other seven swine viruses.The coefficient of variability percent(C/V%)of intro-batch duplicativity test and inter-batch duplicativity test was less 15%.These data demonstrated that the rPDCoV-N-ELISA can be used for epidemiological investigations of PDCoV and that PDCoV had a low serum prevalence in pig population in Heilongjiang province,northeast China.In this study,western blot results showed that the recombinant proteins pET-32a-PDCoV-N and pGEX-6p-1-PDCoV-N can be verified by positive serum of PDCoV,and this rPDCoV-N-ELISA based on recombinant protein antigen pET-32a-PDCoV-N has good sensitivity and specificity,can be a simple and rapid method for detect PDCoV antibodies.