Monoclonal Antibody Preparation,Epitope Identification And Blocking ELISA Establishment Of Porcine Deltacoronavirus N Protein

Porcine deltacoronavirus(PDCoV)is a novel porcine intestinal coronavirus which can cause diarrhea,vomiting and dehydration in piglets.PDCoV was first detected in swine rectal swabs in 2012.Outbreak of PDCoV was reported in 2014 in USA.After that,PDCoV spread to South Korea,Thailand,Vietnam and many provinces of China,which seriously damaged the healthy development of the pig industry.Currently,there is no commercial vaccine and diagnostic reagent of PDCoV in clinical application.Previous studies have shown that the nucleocapsid protein(N protein)of PDCoV can be expressed in large quantities in the early stage of PDCoV infection,and it can induce the body to produce a large number of specific antibodies against the virus,which is the best target protein for PDCoV detection of early infection.In view of this,we prepared a monoclonal antibody against PDCoV N protein,identified the epitope which the antibody react with,and established a PDCoV blocking ELISA antibody detection method based on the antibody and N protein.The specific test contents are as follows.1.Preparation of PDCoV N protein monoclonal antibody.In order to prepare a monoclonal antibody against PDCoV N protein,this assay first performed prokaryotic expression and purification of PDCoV N protein,and identified the immunogenicity of purified N protein by Western blot(WB).Then,mice were immunized with the purified N protein and spleen cells were fused with SP2/0 cells.The positive hybridoma cell lines were screened by indirect enzyme-linked immunosorbent assay(ELISA),the reactivity of monoclonal antibodies to N protein was identified using WB and indirect immunofluorescence assay(IFA),and the subtype of the antibody was identified.Finally,the stability of the antibody produced by the cell lines was evaluated by detecting the antibody levels of different generations of the hybridoma cell lines.The results showed that the purified N protein could react with PDCoV positive serum and had good immunogenicity.Two monoclonal antibodies were screened,which named 6B7 and 7F8,respectively.The the subtype of monoclonal antibody 6B7 is IgG2b while the subtype of 7F8 is IgG2a,both the light chain type is kappa type,and the two hybridoma cell lines could both stably secrete antibodies.The ELISA titer of the monoclonal antibody prepared in this study can reach more than 400,000 times,and can be used for the detection of PDCoV in ELISA,WB and IFA.2.Identification of the epitopes of monoclonal antibodies against PDCoV N protein.To ascertain the epitopes of two monoclonal antibodies against PDCoV N protein,we first analyzed the amino acid sequence of N protein by bioinformatics methods and predicted the epitope region.Then,the N protein was truncated and expressed according to the prediction results.The reactivity between monoclonal antibodies and truncated proteins was analyzed by WB to identify the smallest epitopes of the monoclonal antibodies.The conservation of epitopes between different coronaviruses was identified by homology analysis,and the distribution of antigenic epitopes on N proteins was observed by three-dimensional modeling of N proteins.The results showed that the epitopes targeted by monoclonal antibodies 7F8 and 6B7 were located at amino acids 251-276(NFQAGAITLTFSYSITVKEGSPDYER)and 326333AA(QDWEWDDA)on N protein,respectively.The epitope 251-276AA is an intrinsically disordered conformational epitope and 326-333AA is a linear epitope.Both the two epitopes are highly conservative.The identification of N protein epitope laid a foundation for the study of the structure and function of PDCoV N protein.3.Establishment of PDCoV blocking ELISA antibody detection method.In order to develop a method that can rapidly and accurately detect PDCoV antibodies,the purified N protein and monoclonal antibody 6B7 were used as the coating antigen and detection antibody,respectively.By optimizing the blocking ELISA method,a blocking ELISA antibody detection method for PDCoV was established,and the specificity,sensitivity and reproducibility of this ELISA blocking method were evaluated.The coincidence rate of the method with PDCoV neutralization test was analyzed by the detection of clinical pig serum,and the rabbit or mouse against PDCoV N protein polyclonal antibodies were detected by this method to analyze the application range.The results showed that the blocking ELISA method was coated with 1μg/mL of N protein and detected by enzyme-conjugated antibody diluted 2000 times.The S/N value≤0.559 was determined to be PDCoV positive.The established PDCoV blocking ELISA method was negative for the positive sera of porcine epidemic diarrhea virus,porcine transmissible gastroenteritis virus and porcine sapello virus,which indicated that the method had good specificity.The detection rate of PDCoV positive sera with neutralization titer greater than 8 could reach 100%by this method,which indicated that the method had high sensitivity.This method is stable and had good reproducibility in the same batch and between different batches,has high coincidence rate with virus neutralization test,and can detect antibodies against PDCoV in serum of different species.Therefore this study provides a convenient,rapid and accurate antibody detection method for detecting the infection of PDCoV.In summary,we expressed and purified PDCoV N protein with good immunogenicity,and two monoclonal antibodies 6B7 and 7F8 against N protein were prepared and screened,and the antibody targeted antigenic epitopes were identified.Furthermore,we established a PDCoV blocking ELISA antibody detection method based on the monoclonal antibody 6B7 and N protein.This method has good specificity,high sensitivity and wide application range,which can provide strong technical support for the prevention and control of PDCoV.