Production And Epitope Mapping Of Monoclonal Antibodies Against Swine Acute Diarrhea Syndrome Coronavirus N Protein

Swine acute diarrhea syndrome(SADS)is a novel infectious disease of pig digestive system caused by swine acute diarrhea syndrome coronavirus(SADS-CoV).In early 2017,the outbreak of SADS-CoV was reported for the first time.SADS-CoV-infected pigs have typical symptoms of intestinal infections.The occurrence of SADS-CoV would be a huge problem for the prevention and control of swine diseases,as well as the development of the pig industry.Therefore,it is urgently needed to develop effective vaccines and diagnostic methods for the detection of SADS-CoV infection.Nucleocapsid protein of coronaviruses has varieties of important biological significance.On the one hand,N protein binds viral RNA and is involved in several of the biological activities of the virus;on the other hand,abundant antibodies against N protein could be induced at the early stage of virus infection because of its own high immunogenicity.Such features of the N protein make it an ideal target antigen for early detection and diagnosis of SADS large-scale outbreaks in pig farms.In this study,MAbs against SADS-CoV N protein was prepared,and its application prospects were identified in different detection methods.Firstly,this study has constructed a prokaryotic expression system to express and purify SADS-CoV N protein.And then the concentration was determined.The results of Western Blot and SDS-PAGE analysis suggested that SADS-CoV N protein with high purity was obtained in this experiment,which has a good immunogenicity.Secondly,this study prepared mouse monoclonal antibodies with the purified SADS-CoV N protein.Using indirect ELISA detection method and limiting dilution method,this study finally obtained 5 monoclonal hybridoma cells that can stably secrete anti-SADS-CoV N protein antibodies,namely hybridoma cells 1A5,3E9,4C6,4H6 and 5D7 respectively.Subsequently,ascites antibodies were prepared.The identification of subclasses of these monoclonal antibodies suggested that the light chain of all these five MAbs was?chain.The heavy chain of MAbs 3E9,4C6,as well as 5D7 is Ig G1.The heavy chain of MAb 4H6 is Ig G2a,and that of MAb 1A5 is Ig G2b.The titers of ascites were tested by indirect ELISA method and all of them have good immunogenicity to SADS-CoV at a higher dilution gradient.The result of Western Blot showed that these five MAbs have good reactogenicity and specificity with SADS-CoV,which has no across reactivity with other porcine coronaviruses.Thirdly,the functional application of MAbs was identified in this study.The results showed that MAbs 1A5,3E9,4C6,4H6 and 5D7 can be used in indirect immunofluorescence assay to detect SADS-CoV infected with Vero E6 cells.Further functional identification of MAb 3E9 showed that it reacted positively with the small intestine tissue of SADS-CoV infected piglets by immunohistochemistry assay.MAb 3E9 can also be used to detect SADS-CoV in Vero E6 cells with the method of Co-immunoprecipitation test.Finally,this study mapped SADS-CoV N protein epitopes recognized by MAbs 3E9 and 4C6.With bioinformatics software and the eukaryotic expression system,the results of Western Blot showed that MAbs 3E9 and 4C6 recognized the same linear epitope,namely ³⁴³DAPVFTPAP³⁵¹.Subsequently the amino acid sequence alignment found that the epitope sequence is highly conserved among SADS-CoV isolates and other SADS-related coronaviruses.The results further illustrate that the MAbs 3E9 and 4C6are specific to SADS-CoV N protein.Above all,this study screened out five strains of SADS-CoV N protein hybridoma cells and prepared corresponding ascites.The subtypes,immunogenicity and specificity of these five MAbs were detected.The applications prospects of these MAb 3E9 were analyzed by indirect ELISA,IFA,IHC,as well as Co-IP.Furthermore,the epitopes against N protein recognized by MAbs 3E9 and 4C6 were identified.