Isolation And Identification Of Epitope-specific Antibodies Of Classical Swine Fever Virus Based On Different Methods

Classical swine fever(CSF)is a highly contagious swine disease found worldwide that has caused devastating economic losses.It was listed as a Class A infectious disease by international veterinary organizations.Monoclonal antibodies(m Abs)with neutralizing activity can specifically target and eliminate foreign substances in vivo.In addition,m Abs also play an important role in studying immune mechanisms and developing therapeutic reagents.With the development of technology,various types of m Abs have been obtained which can be used in different fields such as life sciences and biomedicine.However,there are few efficacious monoclonal antibodies against the CSF virus(CSFV)for treatment in vivo,because most m Abs against CSFV are produced by mouse hybridomas can induce the production of anti-mouse antibodies in vivo,leading to a short half-life and the method of producing m Abs from hybridoma cells is usually time consuming and cumbersome.Thus,establishment of a simple,highly efficient,low-cost method to produce anti-CSFV m Abs is necessary for the preparation of diagnostic reagents and antivirus agents for CSFV.The CSFV genome is processed into structural proteins(C,Erns,E1,and E2)and several non-structural proteins by virus-encoded and cellular proteases.E2 protein contains some conformational epitopes and linear epitopes that play critical roles in inducing neutralizing antibodies.In 2000,Lin et al.used the antibody WH303 to identify the epitope TAVSPTTLR by expressing the truncated E2 protein,named epitope 303.This epitope is strongly conserved in CSFV strains but highly divergent among BVDV and BDV strains.The linear epitope was proved to be highly immunogenic and showed potential as candidates for vaccine-related research.Single-cell RNA sequencing(sc RNA-seq)is an emerging technology for accurate quantification of gene expression in individual cells,allowing a more accurate understanding of transcriptomes in individual cells to reveal regulatory relationships between genes and track the trajectories of distinct cell lineages in development.Smartseq2 has the advantages of short time,allowing input of low RNA,and obtaining fulllength c DNA.Therefore,in our study we used this technology to establish a c DNA library of single cells,and extract antibody genes from it.Finally,a platform for efficient antibody preparation was successfully established.In order to establish a method for preparation of epitope-specific antibodies of CSFV E2 protein.(1)An experimental method for identifying antibodies against CSFV E2 protein was established.Firstly,m Abs HK24 and HK44 were expressed in HEK293 T cells,and then we determined that these m Abs can bind to recombinant E2 protein by a series experiments such as Western blot,ELISA,SPR,IFA.Subsequently,molecular simulation docking and amino acid point mutation experiment results showed that the recognition sites of antibodies HK24 and HK44 in CSFV E2 protein within this major antigenic epitope(TAVSPTTLR).In addition,we found that m Abs HK24 and HK44 exhibit potent neutralizing activity against CSFV,and they can protect PK-15 cells from infections in vitro.(2)Synthesis of magnetic nanoparticles capable of specifically separating epitope-specific neutralizing antibodies.In this study,to establish a method for preparation of polyclonal and monoclonal antibodies based on magnetic nanoparticle technology,a magnetic core@ multi-arm shell-epitope(M@A303)bioconjugate was fabricated to enrich and isolate epitope-specific antibodies from immune serum,the antibodies we isolated were named Es Abs303.We determined that epitope-specific antibodies can bind to the recombinant E2 and exhibit potent neutralizing activity against CSFV.(3)A method of preparing an epitope-specific monoclonal antibody was established.In this study,epitope 18-4(Wx EAAYQr FL)that specifically recognizes MCF-7 cells was modified on the surface of magnetic nanoparticles.The capture results of MCF-7 cells showed that magnetic nanoparticles can specifically capture target cells.Thus,we used M@A303 to isolate single epitopespecific cells from peripheral blood mononuclear cells of pigs immunized with attenuated vaccine.In summary,two monoclonal antibodies with neutralizing activity against CSFV E2 protein were successfully obtained,and a method for isolating epitope-specific polyclonal antibodies and monoclonal antibodies was successfully established.This is an efficient and convenient method that provides a new way to prepare antibodies for treatment of human infectious and non-infectious diseases.