Establishment Of Detection Method For Antigen And Antibody Of Porcine Deltacoronavirus

Porcine Deltacoronavirus(PDCoV)is a newly discovered intestinal pathogenic coronavirus.Piglets infected with PDCoV show symptoms such as vomiting,diarrhea and dehydration,as well as high morbidity and mortality,that makes PDCoV one of the important causes of piglet death.At present,PDCoV has been detected not only in many other countries and regions,but also been reported in many provinces in China.In view of the widespread prevalence and potential threat of PDCoV in swine population,this study established indirect ELISA and cellular immunohistochemistry for detection of PDCoV antibody,and double antibody sandwich ELISA for detection of PDCoV antigen.1.Establishment of indirect ELISA for detection of PDCoV antibodyIn this study,the amino acid sequence of PDCoV N protein was analyzed by DNAStar Protean,and the regions with strong antigenicity(265-342 aa)were selected.According to the frequency of E.coli codons,the optimized design was carried out and the corresponding gene fragment N-opti was synthesized artificially.The expression systems of pET-32a and pGEX-6p-1 were used for prokaryotic expression.The obtained fusion proteins were separately labeled with His and GST,named as PDCoV-N-His and PDCoV-N-GST.They are all soluble expressed.The Western Blot results showed that the sizes of the two recombinant proteins were about 31 kDa and 36 kDa,respectively,which were consistent with the expectation.Two recombinant proteins were purified by Ni-NTA affinity chromatography medium and high affinity GST purification medium.The purified recombinant proteins were used as the coating antigen to establish indirect ELISA,comparing two methods,PDCoV-N-His protein as coating antigen is better,finally,an indirect ELISA method for detecting specific antibodies to PDCoV was established using PDCoV-N-His as coating antigen.This indirect ELISA has good specificity,repeatability and stability,since it has no cross-reaction with the positive serum of PEDV,TGEV and PRoV.The variation coefficient of the inter-batch and intra-batch repeatability test was less than 10%.The indirect ELISA was used to detect 20 PDCoV positive serum and 20 negative serum,and the results showed that the positive and negative coincidence rate was both 100%.Our results showed that the indirect ELISA for PDCoV antibodies had good specificity Our results showed that this method had good specificity and could be used for rapid diagnosis and epidemiological investigation of PDCoV infection.2.Establishment of cell immunohistochemical method for detection of PDCoV antibodyPrimers were designed according to the complete PDCoV N gene sequence,and the N gene was cloned by RT-PCR.Then the lentiviral vectors expressing N protein was constructed.The recombinant cell Vero/PDCoV-N with stable expression of N protein was screened by 4 ?g/mL purinomycin.The Western Blot analysis showed that the cell could express 41 kDa protein bands that were consistent with the expected size.Furthermore,a cellular immunohistochemical for detection of specific antibodies to PDCoV was established using this cell.The method was used to detect the PDCoV positive serum,and the cytoplasm showed specific tan after color rendering,while the the control cells showed no color response.This method provides another effective serological method for the rapid diagnosis and epidemiological investigation of PDCoV infection.3.Establishment of double antibody sandwich ELISA detection method based on PDCoV N protein monoclonal antibodyIn this study,PDCoV-N-His protein was used as the immunogen,PDCoV-N-GST protein was used as the detection antigen.Six hybridoma cell lines capable of stable secretion of PDCoV N protein monoclonal antibody were obtained by ELISA,which were named 1A2,4C5,5C5,8C12,8E8 and 10A7,respectively.The results of subclass identification showed that four monoclonal antibodies were IgGi subtype and the two were IgM subtype.After analysis of the relative affinity and antigen-binding epitopes of 6 monoclonal antibodies,1A2 was selected as the capture antibody and HRP-conjugated 5C5 as the detection antibody,and a double antibody sandwich ELISA was established.Linear correlation coefficient of the method's double logarithmic regression fitting was greater than 0.99,and detecting range of quantitative analysis for PDCoV N protein was 54 ng/mL to 1.3 ?g/mL determined by regression equation.The coefficient of variation of the inter-batch and intra-batch repeatability test was less than 10%,indicating that the method had good repeatability and stability.The double monoclonal antibody sandwich ELISA showed an obviously detection effect on recombinant N protein,and it is expected to discover a further development of the specific antigen detection method for clinical PDCoV infection diagnosis.