Identification And Application Of Epitope Recognize By Monoclonal Antibody Against Porcine Deltacoronavirus N Protein And Preparation Antibody Against S1 Protein And Its Related Receptors

Porcine Deltacoronavirus（PDCoV）is a newly discovered porcine intestinal coronavirus that causes vomiting,dehydration,and watery diarrhea in piglets.It is highly pathogenic to piglets and poses a great threat to the pig industry.Porcine Deltacoronavirus mainly contains four structural proteins,namely,Spike（S）protein,Nucleocapsid（N）protein,Membrane（M）protein and Envelope（E）protein.Among them,the N protein was the most conservative,and it was most abundant in the infected host cells,showing strong conservation among different strains,thus it was often used as a target protein for clinical detection.Spike protein exists on the cell surface and is divided into two subunits,S1 and S2.The S1 subunit contains the main site for stimulating the host to produce neutralizing antibodies as well as antigen-antibody binding sites.The identification of antigenic epitope plays an important role in the subsequent screening of the core epitope,dominant epitope and vaccine development.Therefore,in this study,the laboratory-prepared monoclonal antibody4D3 against PDCoV N protein was subjected to accurate epitope identification.A double antibody sandwich ELISA based on PDCoV N protein was established by using monoclonal antibody 4D3 and rabbit-derived polyclonal antibody against N protein,which enriched the clinical detection methods of PDCoV antigen.At the same time,the PDCoV S1 protein as well as three host proteins including Angiotensin Converting Enzyme 2（ACE2）,Aminopeptidase N（APN）and Transferrin Receptor（TfR）were successfully expressed,and the corresponding polyclonal antibodies were prepared.Furthermore,whether the three host proteins are the receptors of PDCoV was preliminarily identified.The specific research contents are as follows:1.Identification of the B cell epitope of Porcine Deltacoronavirus N proteinIn order to accurately identify the epitope recognized by the monoclonal antibody 4D3against the PDCoV N protein,based on the prediction result of the online epitope prediction website,we performed five rounds of truncation on the PDCoV N protein,cloned the fragments into p ET-32a and PGEX-4T-1 vectors,converted into E.coli BL21（DE3）for expression,and identified by SDS-PAGE.After analysis by Western Blot,the exact epitope of PDCoV N protein was finally determined to be ²⁸QFRGNGVPLNSAIKPVE⁴⁴.Further comparative homology analysis of the identified B cell epitope（aa28-44）revealed that the epitope was relatively conservative among PDCoV strains,and only two types of amino acid residues,valine（V）and alanine（A）,existed at position 43.Of these,V at position 43 was found in the US,Japan and South Korea lineages,while the position was A in early Chinese lineages.Three-dimensional structure modeling of N protein showed that the epitope structure we identified had typical B cell epitope characteristics.The identification of the precise epitope of the PDCoV N protein enriches the epitope library of the N protein and lays a foundation for the development of diagnostic reagents and vaccines related to PDCoV epitope and subsequent research.2.Establishment of the monoclonal antibody sandwich ELISA for detection of Porcine DeltacoronavirusIn this study,the purified monoclonal antibody 4D3 was used as the coating antibody,and the HRP-labeled polyclonal antibody against PDCoV N protein was used as the detection antibody.Through the optimization of experimental conditions,a sandwich ELISA detection method based on porcine deltacoronavirus N protein was successfully established.The optimal experimental conditions after optimization were as follows:the optimal coating concentration of monoclonal antibody 4D3 was 2μg/m L,the optimal dilution of detection antibody was 1:2000;and the optimal coating time was incubation at 4℃overnight.The best blocking solution was 5%BSA,the samples were incubated at 37℃for 1 h;and the action condition of the enzyme-labeled antibody detection was incubation at 37℃for 1 h.The specificity experiment showed that the sandwich ELISA developed in this study did not cross-react with PEDV and TGEV,and had good specificity.The sensitivity test results showed that the minimum detectable amount of PDCoV by this method was 10³TCID₅₀/m L,and the minimum detectable amount of PDCoV N protein was 31.25 ng/m L.The coefficient of variation was less than 5%both within and between batches.It indicated that the sandwich ELISA based on PDCoV N protein was successfully established in this study,and it had a broad prospect of clinical application.3.Preparation of antibodies against Porcine Deltacoronavirus S1 protein and preliminary identification of related suspected receptorsIn this study,the PDCoV S1 gene and three pig-derived host proteins（ACE2,APN and TfR）were amplified by RT-PCR,cloned into p ET-32a vector,sequenced correctly,and transferred to E.coli BL21（DE3）competent cell for expression.After the optimal expression condition was determined,a large number of proteins were expressed.After SDS-PAGE,Coomassie brilliant blue staining analysis showed that the four membrane proteins mainly existed as inclusion bodies.The recombinant S1 protein was purified by Ni column,and the three recombinant host proteins,ACE2,APN and TfR,were purified by glue cutting.The purified protein was subjected to gradient dialysis renaturation.Western Blot results showed that the four recombinant proteins had good reactivity with the Anti-His antibody,indicating that we had successfully prepared the PDCoV S1 protein with good antigenicity together with ACE2,APN,and TfR proteins through prokaryotic expression.After the renatured PDCoV S1 protein as well as the three recombinant host proteins ACE2,APN,and TfR were mixed and emulsified with the adjuvant,the BALB/c mice aged from 6 to 8 weeks were immunized.The blood was collected from the orbit of mice with serum titer up to 10⁵,and the separated serum was the polyclonal antibody.The obtained polyclonal antibodies were identified by Western Blot and IFA,and the results showed that the prepared S1 antibody could react with the full-length S protein and S1 protein of the virus,and the polyclonal antibodies against ACE2,APN and TfR could all react with their corresponding natural proteins.After blocking the corresponding host protein on LLC-PK1 with antibodies against ACE2,APN and TfR,PDCoV was inoculated for IFA identification and titer determination.The results showed that blocking of APN had a significant effect on PDCoV infection,while blocking of ACE2 and TfR had no significant effect on PDCoV infection,preliminarily indicating that APN might be a suspected receptor of PDCoV.