Identification Of Porcine Deltacoronavirus N Protein Epitope And Development Of An Indirect ELISA Method Based On The Epitope

Porcine deltacoronavirus(PDCoV),also known as Porcinedcoronavirus,was first reported in Hong Kong,China in 2012,and then spread all over the world,posing a threat to the pig industry.Because the clinical manifestations of pigs infected with PDCoV are similar to those of other pig gastrointestinal diseases,the clinical diagnosis becomes complicated.Therefore,it is very important to establish an effective diagnosis method of PDCoV.In this study,five hybridoma cells were prepared by immunizing BALB/c mice with PDCoV N protein expressed and purified in prokaryote.One hybridoma cell line 4E88 was screened from the hybridoma cells for monoclonal antibody preparation and epitope identification.An indirect ELISA method was established for the detection of epitope peptides.1.Preparation and identification of monoclonal antibodies against PDCoV N proteinIn this study,PDCoV N protein was expressed and purified by p ET-28a-N expression bacteria,which was successfully constructed in the early stage.Then,BALB/c mice were immunized to prepare hybridoma cells.Five hybridoma cells(11E,7D2,4E88,6A5 and 3B5)were screened out.The five hybridoma cells were identified by indirect ELISA and indirect immunofluorescence assay(IFA).One hybridoma cell line 4E88 with better antigen reactivity was selected for antibody preparation and identification.The characteristics of monoclonal antibody 4E88 were identified by Western blot and subtype identification.The application value of monoclonal antibody 4E88 was identified by immunohistochemistry,flow cytometry and tissue fluorescence detection.The results showed that the monoclonal antibody4E88 belonged to Ig G1 subtype and kappa chain;the Western blot showed that 4E88could specifically recognize the virus N protein and fusion N protein of PDCoV;the application of the monoclonal antibody in flow cytometry,immunohistochemistry,tissue paraffin section fluorescence test to detect the virus was better.2.Epitope identification of N-protein monoclonal antibody 4E88The monoclonal antibody 4E88 was used to search for its epitope.After reacting with a series of truncated proteins,the epitope recognized by the monoclonal antibody was located at aa308-AKPKQQKKPKK-318.After further reducing the amino acids at the left and right ends,it was found that the minimum epitope recognized by the monoclonal antibody 4E88 was 309-KPKQQKKPK-317(named EP-4E88).The homology analysis showed that EP-4E88 was highly conserved in 25 PDCoV strains,which was 100%.It was highly similar to Sparrow coronavirus(HKU17),Asian leopard cat coronavirus(ALCCoV),Quail coronavirus(UAE-HKU30)and Sparrow triangle coronavirus(Sp DCoV),which was more than 90%.However,the similarity of EP-4E88 to other porcine coronaviruses is less than 22.2%,indicating that EP-4E88 has the potential to distinguish PDCoV from other porcine coronaviruses.3.Establishment of indirect ELISA based on EP-4E88An indirect ELISA method based on EP-4E88 epitope was established to optimize the reaction conditions.The concentration of coated antigen was0.5mg/100mL,the dilution of serum was 1:50,and the dilution of secondary antibody was 1:5 000.The critical value criterion is positive when OD₄₅₀³0.262.The positive sera in 7 kinds of porcine virus ELISA kits,including CSFV,PRRS and PCV2,were used to detect the established epitope indirect ELISA,and the results were negative;In the sensitivity test,the highest dilution of the positive serum detected by ELISA was 1:3200,and the repeatability test showed that the repeatability within and between batches was better,less than 10%.A total of 471 clinical sera from 23 regions in Sichuan Province and Qianjiang region in Chongqing were detected by the established epitope indirect ELISA.The results showed that the positive rate of PDCoV was 9.5%.It mainly occurs in Xichang,Tongjiang and Nanbu counties of Sichuan and Qianjiang of Chongqing.