Preparation Of Anti-Phenylethanolamine A Single-Chain Antibody And Preliminary Study On Its Molecular Recognition Mechanism

β-stimulant substitute is often used as a growth-promoting agent to affect the redistribution of nutrients in animals,increasing the rate of lean animal ketone bodies,and increasing the feed conversion rate.However,the residues of such drugs in the edible tissues of animals（lung,liver,kidney and muscle）will threaten the health of consumers through the food chain.China and the European Union have classified it as a"zero tolerance"drug and stipulated that beta-stimulant prototypes,their salts,esters and other derivatives should not be detected in any organization.However,driven by interests,the illegal use ofβ-stimulants still exists.Phenylethanolamine A was detected in feed for the first time in China in March 2011,which attracted people’s attention regarding its residual hazards in edible tissues.Thence,it is urgent to develop a method for the detection of phenylethanolamine A residues.The immunoassay method has been widely used in the detection of phenylethanolamine A residues,due to high sensitivity,high throughput,easy operation,and on-site.As we all know,the core reagent of immunoassay is antibody which performance plays an important role in the sensitivity of immunoassay method.In recent years,recombinant antibodies produced by recombinant DNA technology and antibody engineering technology have developed rapidly.In recent years,recombinant antibodies have provided a new idea for antibody preparation due to their short cycle,strong operability.Antibody library technology can effectively realize the connection between genotype and phenotype.The development of this technology replaces hybridoma technology,avoids animal immunity,greatly shortens the antibody preparation cycle and simplifies operations.Among them,phage display technology is the most mature antibody display tools.By optimizing screening and elution strategies,it is possible to obtain recombinant antibodies and gene sequences with excellent characteristics.Subsequently,based on the known nucleotide and amino acid sequences combined with modern computer simulation technology,new antibody molecules with ideal characteristics are prepareded to complete the evolution of antibodies in vitro,thereby further promoting the development of immunoassay technology.A single-chain antibody was prepared to complete the methodological study of phage antibody surface display technology,which provided a new recognition element for immunoassay methods.The anti-phenylethanolamine A single-chain antibody provided a good material for in vitro evolution research and recognition mechanism research.The research contents and results are as follows:（1）The hybridoma cell line（PEAA/2D8）were used to construct a phage antibody library with a library capacity of 8×10⁸ using phage antibody display technology.Four rounds of screening were carried on the phage antibody library,and finally a single chain antibody（PEAA-scFv-4-13）was obtained.In this process,the concentration of PEAA-NH₂-OVA in each round was gradually reduced,and the washing times of PBST were gradually increased.The elution strategy for specific antibodies was to combine acid elution and competitive elution.By optimizing the induced expression conditions（30℃,200r/min,0.1 m M IPTG）,PEAA-scFv-4-13 was prokaryotically expressed to achieve soluble expression.（2）The immune activity of PEAA-scFv-4-13 was studied.Using PEAA-NH₂-OVA as the coating agent and phenethanolamine A as the competitor,ic-ELISA was performed.The result showed that the IC₅₀ was 16.98μg/L,which was much higher than its parental monoclonal antibody（0.63μg/L）;With salbutamol,clenbuterol and ractopamine as competitors,ic-ELISA was performed to determine the IC₅₀ value of each drug,and the cross reactivity（CR）was calculated.The results showed that PEAA-scFv-4-13 has strong specificity.It basically did not recognize otherβ-agonists,which was consistent with its parental monoclonal antibody.PEAA-scFv-4-13 diluted to working concentration was stored at 4°C and 37°C,respectively.Samples were taken on days 0,2,8,14 and 21 for indirect ELISA.The antibody stability was evaluated by the change of the OD value of“0”well in different days.The activity of PEAA-scFv-4-13 stored at 4°C gradually decreases and can be maintained for about 2 weeks;The antibody activity of PEAA-scFv-4-13 stored at 37°C decreased by 50%on the second day.In summary,the affinity and specificity of PEAA-scFv-4-13 are similar to the parent monoclonal antibody,but the stability is far less than that of the monoclonal antibody.（3）This study verified the key amino acid sites of the interaction between PEAA-scFv-4-13 and phenylethanolamine A.Firstly,a model protein with a homology of 56.05%was constructed through SWISS-MODEL,and the model was appraised exploiting UCLA-DOE LAB-Laboratory Services.The results showed that the quality of the model was good.The docking model of PEAA-scFv-4-13 and Phenethanolamine A was constructed virtually with Sybyl X-2.0 docking software.The lowest energy docking model was selected from the generated 50 docking models,and its binding energy was-6.6 kcal/mol.Through the analysis of the docking model,it was known that F103 and E174 may play a role in the recognition of phenylethanolamine A and affect the affinity of the antibody.Subsequently,PEAA-scFv-4-13 protein mutants（F103-A,E174A）were obtained by alanine scanning mutation technology to explore the influence of these two amino acid positions on antibody affinity.Indirect ELISA was performed through PEAA-scFv-4-13 prototype and its mutant respectively as immune elements.By comparing the titers of the three scFvs,it was verified whether the amino acid at this site was a"hot spot"amino acid.The results showed that the titers of the two F103A and E174A mutants were lower than PEAA-scFv-4-13,indicating that the F103 and E174 amino acid positions had a certain contribution to the affinity of PEAA-scFv-4-13.Finally,the analysis of the docking results showed that the scFv active pocket was mainly composed of VHCDR₃,VHFR₄,VLFR₂,and VLFR₃,and a total of 13amino acids were directly involved in the formation of the active pocket.The main forces between PEAA-scFv-4-13 and Phenethanolamine A were hydrogen bonds and intermolecular forces.In this study,the methodology of phage antibody surface display technology was completed,and a single-chain antibody against PEAA（PEAA-scFv-4-13）was successfully prepared to provide a new type of antibody for immunoassay methods.The recognition mechanism between PEAA-scFv-4-13 and phenylethanolamine A was analyzed with PEAA-scFv-4-13,which laid the foundation for in vitro maturation research of antibodies.This study provided a new research idea for the rapid detection method of phenylethanolamine A in edible tissues of animals.