Preparation Of Full-human Anti-RT Single-chain Antibody And Study On Its Biological Activity

Ricin?RT?is a phytotoxin extracted from castor bean.It is a type II ribosomal activity inhibitor and contains A subunit?RTA?and B subunit?RTB?.Ricin has the characteristics of low preparation cost,stable properties,multiple poisoning pathways,and no specific antidote.As a biological warfare agent and terrorist agent,ricin is a serious threat to public safety.At present,ricin does not have any antidote,and specific antibody therapy may become an effective treatment.Single chain antibody?sc Fv?has the advantages of high affinity and high specificity of the parent monoclonal antibody to the antigen,and also has many advantages such as low immunogenicity in the body and easy penetration of the barrier tissue in the body.Using phage antibody library technology,the single-chain antibody gene is recombined to a specific position of the phagemid,and the single-chain antibody is fused and expressed with the phage coat protein to finally obtain the phage antibody.Not only does it avoid cumbersome immunization and cell manipulation,but it is also easy to modify the antibody,which is beneficial to improve the affinity of the antibody.This study explored the preparation of fully human single-chain antibodies specific for ricin,mainly including the following four parts:1.Screen antibody library to prepare fully human anti-RT single chain antibodyIn this study,through the four rounds of"adsorption-washing-elution-amplification"enrichment screening of the Tomlinson I+J phage antibody library,we successfully screened and obtained the fully human anti-ricin toxin single chain antibody JE11.Through PCR and phage ELISA antigen cross-reaction,the positive clones obtained were further identified,and clone JE11 with high positive rate and low false positive rate was screened.The primers were designed to perform Ig BLAST on the screened JE11 sequence,and it was proved that the single-chain antibody is a human antibody,and its VH belongs to VHIII subgroup,and VL belongs to?chain,which belongs to V??subgroup.2.Study on the expression process of fully human anti-RT single chain antibodyIn order to determine the optimal expression process for the preparation of fully human anti-RT single-chain antibodies,this study compared the expression of single-chain antibodies under different expression conditions by SDS-PAGE and grayscale analysis to determine the optimal induced expression of JE11 engineering bacteria The conditions were:induction at 37?for 12 h at 0.5 mmol/L IPTG concentration.The single-chain antibody crude extract was purified by metal chelation chromatography and ion exchange chromatography,and the purity of the product was analyzed by thin layer scanning.The purification method of the inclusion body of JE11was finally determined as follows:the metal chelate affinity layer was used first The protein eluted with 50 mmol/L imidazole and the target protein with 100 mmol/L imidazole.Then,the target protein is passed through an anion exchange chromatography column with a p H=7.2 equilibration solution,and the target protein flows out in the form of flow-through.This study obtained 97.88%pure JE11 antibody,which laid the foundation for the next step in vivo and in vitro experiments.3.Biological activity of fully human anti-RT single chain antibodyIn this study,the ELISA method was used to verify the dose-dependent specific binding of human anti-RT single-chain antibody JE11 to RT,and the linearity rate R²reached 95.23%.In addition,the Western Blot experiment also verified the specific binding of human anti-RT single chain antibody JE11 to RT.It was verified by MTT that 10?g/m L JE11 can protect He La cells against the toxicity of 10×IC₅₀RT,and intramuscular injection of JE11 and RT to mice also proved that 320?g JE11 per mouse could protect mice from 2×LD₅₀RT within 72 h.4.Application of bioinformatics technology to improve antibody bindingIn this study,the molecular model of RT and JE11 was docked through Discovery Studio software,and a composite model of RT and JE11 was obtained.Since most of the amino acids in the binding interface are concentrated in the heavy chain variable region,a single point mutation strategy is adopted to mutate the amino acids on the CDR region of the heavy chain variable region or the binding interface to twenty natural amino acids.Through software combination and affinity comparison,three mutants were finally designed for the next experiment.The ELISA method was used to compare the affinity difference between the three JE11 mutants and JE11.ELISA results showed that the affinity and stability of JE11-5759 were significantly improved.In summary,this study prepared fully human anti-ricin toxin single-chain antibodies by screening a library of fully human phage single-chain antibodies,and carried out studies on the expression conditions,purification process optimization and biological activity of single-chain antibodies.The application of bioinformatics technology improves the stability and affinity of the antibody.The fully humanized anti-ricin toxin single chain antibody prepared by the study has laid a solid application foundation for the detection of ricin and the diagnosis or treatment of ricin poisoning.