| Botulinum toxin is a highly toxic protein neurotoxin produced by anaerobic Clostridium botulinum.It is currently the most toxic substance known in nature and biological and chemical toxins.There are seven serotypes（A to G）in BoNT.Naturally,A,B,E,and F can cause human botulism.Among them,botulinum toxin type A is highly toxic and has many reported cases,leading to more studies on its monoclonal antibodies.Monoclonal antibodies to botulinum toxin type B are not as extensively studied as type A.To date,no effective neutralizing antibodies have been used for clinical use.Therefore,it is of great significance to screen monoclonal antibodies against botulinum toxin type B.The purpose of this study was to screen monoclonal antibodies with high specificity and affinity for botulinum toxin type B through B cell hybridoma and phage antibody library technology.Using hybridoma technology to immunize BALB/c mice with protein BoNT/BHc as the antigen,take the mouse tail spleen cells with the highest titer and fuse with Sp2/O myeloma cells,with an average fusion rate of 95%and an average positive rate 84%.After three cloning cultures by limiting dilution method,nine hybridoma cell lines capable of stably expressing antibodies were obtained.Positive cell lines 1E9-H2 with good growth status and high titer were selected to prepare ascites antibodies.After purification by Protein G affinity chromatography,the purity of the antibody was greater than 96%,and the molecular weight was consistent with the size of the light chain of the heavy chain of the antibody.The linear epitope specifically bound to the antigen BoNT/BHc.According to the subtype identification,the heavy chain of the antibody is IgG1 type and the light chain isκtype,which are all the common subtypes in mouse monoclonal antibodies.The affinity constant of 1E9-H2 antibody to BoNT/BHc antigen is 6.7×10-9mol/L,which has reached the n M level.It is a mouse monoclonal antibody with high affinity.Four antigens,BoNT/AHc,BoNT/BHc,BoNT/EHc and BoNT/FHc,were used to co-immunize mice to obtain a broad-spectrum antibody with neutralizing activity against botulinum toxin type A,B,E and F.After combined immunization,the titers of the four antigens reached 1:32000.The most potent mouse spleen cells were fused with Sp2/O myeloma cells,and the fusion rate was 100%.Among them,the positive rate of cells that can produce anti-A,B,E and F botulinum toxin broad-spectrum monoclonal antibodies is 21.09%.From these,14 positive cells were selected for 3 times of clonal culture using the limiting dilution method,and 4 positive cells of 2B11,3H3,1C5,and 3C3 were selected to prepare ascites antibodies.After protein G column purification,the antibody purity is greater than 90%,and the molecular weight of the antibody is consistent with the size of the heavy and light chains.After non-competitive ELISA,both antibodies can cross-react with the four antigens and have a broad spectrum.The affinity constant of antibody 2B11 for the four antigens of BoNT/A/B/E/FHc is 2.8×10-8,2.8×10-8,2.2×10-8,2.6×10-8mol/L;The affinity constants of antibody 3H3 for the four antigens BoNT/A/B/E/FHc are 2.5×10-8,1.2×10-9,3.8×10-8,3.4×10-8mol/L;The affinity constants of antibody 1C5 for the four antigens BoNT/A/B/E/FHc are3.7×10-8,8.8×10-9,5.0×10-8,5.9×10-8mol/L;The affinity constants of antibody 3C3 for the four antigens BoNT/A/B/E/FHc are 6.7×10-8,1.4×10-9,6.9×10-8,8.8×10-8mol/L.Among them,antibodies 3H3,1C5 and 3C3 have the highest affinity for BoNT/BHc among the four antigens,reaching n M level.The subtype identified that the heavy chains of 2B11,1C5 and 3C3antibodies were of IgG1 type and the heavy chain of 3H3 antibody was of IgM type.The light chains are allκtype.The 2B11,3H3,1C5 and 3C3 antibodies have been identified as high-affinity mouse broad-spectrum monoclonal antibodies.In addition,phage display technology was used to construct a single-chain antibody library of murine phage containing a full set of antibody variable region gene information.BALB/c mice were immunized with the BoNT/BHc protein as the antigen,and mouse spleen cells with a serum titer of 1:64000 were selected as the gene source.The extracted total RNA was reverse-transcribed into cDNA and the variable regions of antibody light and heavy chains were amplified by PCR.Genes were randomly assembled into a single-chain antibody fragment ScFv,which was ligated into the phagemid vector pCANTAB-5E using molecular cloning.The insertion rate of ScFv was 90%identified by PCR.A phage single with a library capacity of 5.0×105was successfully constructed Chain antibody library.BoNT/BHc protein was used as the antigen to solid-phase screening the phage antibody library for four rounds of adsorption-elution-enrichment.Two mouse-derived single-chain antibodies ScFv-A9 and ScFv-A1 were successfully selected.The humanized antibody was humanized to construct a chimeric antibody expression vector.The mammalian expression system Free Style TM 293-F was used to successfully express the humanized chimeric antibody ScFv-A9.Purified and obtained 99%high-purity humanized antibody.protein.In summary,a total of 6 monoclonal antibodies against botulinum toxin type B were obtained after screening,including 5 murine antibodies and 1 human antibody.Among them,2B11,3H3,1C5,and 3C3 antibodies are broad-spectrum monoclonal antibodies that can cross-react with Hc segments of botulinum toxins of types A,B,E,and F.The monoclonal antibodies obtained in this study laid the foundation for the development and testing of botulinum toxin antibody drugs.The constructed antibody library can be used for mass screening of botulinum toxin type B monoclonal antibody. |
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