| Objective: Botulinum neurotoxin is a potential biological warfare agent that poses a great threat to national defense security and people’s health due to its super-toxicity,rapidacting and difficult to prevent characteristics.Since the main anti-toxic strategy research and key technology programs for botulinum neurotoxin have been dominated and controlled by the United States and other Western countries,it is a stranglehold key technology.Domestic and military research on this type of toxin has not made the desired progress,and the antitoxic strategy is not yet mature.Existing studies have shown that antitoxin-neutralizing monoclonal antibodies can effectively neutralize different serotypes of botulinum neurotoxins in vitro,but there are still the following problems in applying them in vivo:(1)monoclonal antibodies have a short circulating half-life in living organisms,making it difficult to exert long-term protective effects;(2)monoclonal antibodies are difficult to enter the intracellular area;(3)botulinum toxin invades very quickly,and the existing monoclonal antibodies are applied in a way that cannot meet the extremely demanding immediacy requirements.These problems have led to the fact that the in vivo antitoxic efficacy of antitoxin monoclonal antibodies has never been widely recognized,and the protective strategies that should be used need to be further explored.This is a key scientific question in the field of botulinum neurotoxin monoclonal antibody control research.Therefore,in this study,we proposed a novel passive immunoprophylactic antitoxin strategy of "pre-administration","intracellular synthesis" and "long-term expression" to address this scientific problem,and developed an engineering scheme using adenoassociated virus as the carrier and single-chain antibody as the antitoxin effector.Methods: Recombinant expression and purification of Clostridium botulinum neurotoxin heavy chain(BONT/AHc)gene.The amino acid and DNA sequence of BONT/A carboxy-terminal receptor binding domain was identified by GENEBANK and Uniprot database search as the natural template,and its sequence was optimized and cloned into p GEX-6P-1 vector by reducing the rare codon content and preferred codon substitution.The plasmid was transfected with E.coli BL21(DE3),and the expression of BONT/AHc antigen protein was induced by Isopropylβ-D-Thiogalactoside(IPTG),and the target protein was extracted,enriched and purified by GST affinity chromatography and AKTA pure purification system.The sequence and purity were verified by protein profiling and SDS-PAGE.BONT/AHc monoclonal antibody screening and identification and Single-Chain Fragment Variable(sc Fv)recombinant preparationBALB/C mice were immunized with three rounds of antigen adjuvant mixture and impulsive immune using purified BONT/AHc protein as the antigen,and the antibody titer of mouse tail serum was detected by ELISA.The immunized mice were subjected to preattack assay,and the best protected immunized mice was selected.Its spleen lymphocytes were extracted and prepared under aseptic conditions and mixed with pre-prepared mouse SP2/0 myeloma cells,and hybridoma cells were prepared by PEG method.Antibody titer of cell culture supernatant was detected by ELISA.The hybridoma cell lines were subcloned by limited dilution method.The amino acid sequence of the variable region of the monoclonal antibody was obtained by hybridoma sequencing,and sc Fv was constructed by ligation with a simple linker(GGGGGGSGGGGSGGGGS)and cloned into p GEX-6P-1 and p ET-22b(+)expression vectors to explore the optimal prokaryotic expression pattern of sc Fv in E.coli.The recombinant proteins were extracted,enriched and purified by nickel column affinity chromatography and AKTA pure system,and then their sequences and purity were verified by protein profiling and SDS-PAGE.Characterization of single chain antibody properties and vectored immunoprophylaxis(VIP)strategyBinding between single chain antibodies and BONT/AHc antigens was verified using western blot.Furtherly,the affinity of sc Fv to BONT/AHc was detected by SPR technique in BIAcore.The sc Fv gene was cloned into the AAV vector to establish a virus-mediated VIP antiviral strategy.The purified virus was diluted to a predetermined volumetric dose and injected into the gastrocnemius region of mice.Serum antibody concentration was determined by ELISA,and a defined dose of Bo NT/A toxin was injected intraperitoneally into mice during the peak antibody period to establish a mouse attack model.Mice were observed to survive for 14 consecutive days,and the daily number of deaths in each group was recorded and survival curves were plotted to assess the intracellular antibody neutralizing activity and protection in vivo.The intracellular antibodies were neutralized and protected in vivo.The best scored model was selected by Alphafold2 and the protein structure was verified by SAVES 6 and Rasch diagram.Protein-protein molecular docking was performed,uploaded to PDBe PISA to analyze the interaction surface in the protein complex,analyze the antigen-antibody complex interaction mechanism,and visualize it using pymol software.Results: The BONT/AHc-p GEX-6P-1 prokaryotic expression plasmid was constructed,and the high purity BONT/AHc recombinant protein was obtained and endotoxin and tag were removed using E.coli prokaryotic expression system,GST affinity chromatography and AKTA PURE purification system,and Pre Scission protease.The size of the recombinant BONT/AHc antigen protein was identified by SDS-PAGE to be about 45 k Da,and the purity was higher than 95%,meeting the requirements of immunized animals.Six BALB/C mice were immunized using BONT/AHc protein,and the results of ELISA of mouse tail serum showed that the serum antibody titers of the remaining four immunized mice were higher than 1:200,000 except for immunized animals 84 and 86,and mouse 82 had the highest antibody potency level.After the pre-attack assay,the hybridoma cell line was established and subcloned with mouse 82 spleen lymphocytes and mouse SP2/0myeloma cells,and finally a monoclonal hybridoma cell line with 100% positive rate was identified,named 82-G10,the secreted monoclonal antibody of which had strong binding activity to BONT/AHc.The 82-G10 hybridoma cells were sequenced,VH and VL sequences were extracted and the complete 82-sc Fv gene sequence was constructed by simple linker(GGGGGGGGGSGGGGGGGS)ligation.The 82-sc Fv plasmids were constructed using PGEX-6P-1 and p ET-22b(+)vectors respectively,and the feasibility of p ET-22b(+)vector was confirmed and the soluble expression of 82-sc Fv was achieved in E.coli prokaryotic expression platform,which was enriched and purified by nickel column affinity chromatography and AKTA PURE.The size of 82-sc Fv recombinant protein was about25 k Da,and the purity was above 95%.The binding of 82-sc Fv to BONT/AHc was confirmed by protein immunoblot identification and further assessed by surface plasmon resonance(SPR)technique in Biacore for the binding kinetics of 82-sc Fv single-chain antibody to BONT/AHc antigen with a KD value of 1.148E-09.Based on 82-sc Fv to establish an AAV vector-mediated Immunoprophylactic antiviral strategy was established based on 82-sc Fv,and serum antibodies reached a peak of approximately 70μg/ml around day 10 by intramuscular viral injection.mouse attack assays showed that the 82-sc Fv treated group exhibited a nearly doubled trend of improved survival compared to the control group.Predictive modeling of the three-dimensional structure of the antigen-antibody complex was performed using Alpha Fold2,and the interaction mechanism was analyzed and visualized.Conclusions: This work addresses the key scientific issues in the field of botulinum neurotoxin monoclonal antibodies prevention and control research,and innovatively proposes a new passive immunoprophylactic antitoxin strategy against botulinum toxin.Meantime,we develops and establishes an engineering scheme using adeno-associated virus as the carrier and single-chain antibody as the antitoxin effector,achieves the expected purpose of this new passive immunoprophylactic antitoxin strategy in the in vivo prevention and control of Clostridium botulinum neurotoxin type A "pre-administration" "intracellular synthesis" "long-term expression",forms an antitoxin reserve capacity against botulinum toxin A,and helps solve the "neck" problem in national defense security,which is reflected in:1.Using purified BONT/AHc protein prepared from protoplasm as antigen to immunize BALB/C mice,we innovatively adopted the mouse pre-attack experiment model and optimized the antitoxin neutralizing monoclonal antibody screening protocol,which changed the conventional research paradigm from "antibody screening first-neutralization study later" to "pre-neutralization to determine the effect-antibody screening-neutralization validation again".As a result,the hybridoma cell line 82-G10,which secreted monoclonal antibodies,was selected by the hybridoma assay,and its secreted monoclonal antibodies not only had better binding activity to BONT/AHc,but also its neutralization efficacy was pre-validated.2.We explored the prokaryotic expression and preparation of sc Fv single-chain antibody,and achieved the soluble and high-purity expression of 82-sc Fv single-chain antibody.82-sc Fv was able to bind specifically to BONT/AHc and had excellent affinity activity.Meanwhile,the 3D structure prediction modeling of the antigen-antibody complex by Alpha Fold2 provides structural biological support for its interaction analysis.3.We have creatively explored and established a novel VIP strategy for toxin protection,namely AAV-mediated neutralizing intracellular antibody presentation scheme,which has expanded the application scope of VIP strategy.The AAV-mediated antibody genetically engineered drμg was successfully constructed based on 82-sc Fv.In the mouse attack model established by intraperitoneal injection of botulinum toxin type A,the AAV-mediated VIP strategy effectively reduced the mortality rate of mice exposed to botulinum toxin type A in the prophylactic mode,proving its protective role and immunopreventive value in response to toxin attack. |
PDF Full Text Request