Screening And Evolution Of Enrofloxacin Single-chain Antibody And Establishment Of Detection Method

Enrofloxacin（ENR）is a veterinary drug specific antibiotic,which is widely used in the treatment of bacterial diseases in cultured animals.However,the use of veterinary drugs is often not standardized in clinical practice,which leads to the excessive residue of ENR in cultured animals and the accumulation of ENR in human body through food chain.When it exceeds a certain limit,it will cause serious damage to multiple systems and organs of human body.Although monoclonal antibodies have been used to detect the content of ENR,traditional monoclonal antibodies have some defects,such as non evolution and complicated production process.Therefore,it is necessary to develop a rapid detection method for ENR based on a new antibody.ObjectiveThe purpose of this study is to screen high specific single chain variable fragment antibody（Sc Fv）of ENR by constructing a large library of immune phage Sc Fv,and then use computer simulation and site directed mutagenesis technology to improve the sensitivity of Sc Fv evolution.Based on the Sc Fv mutant,a more accurate and efficient new detection method is established for the detection of ENR residues in animal food.Methods1.The complete antigen was prepared by coupling ENR with carrier protein by carbodiimide method.The complete antigen was used to immunize mice.Spleen tissues were collected.The heavy chain and light chain of single chain antibody were amplified by total RNA extraction,reverse transcription c DNA and PCR.Then the heavy chain and light chain were linked by linker by overlap extension PCR to assemble single chain antibody gene.2.Phage display technology was used to connect Sc Fv gene with phage vector pcantab5e to prepare recombinant vector,which was transformed into host strain TG1to construct phage antibody library.After several rounds of panning and enrichment,the monoclonal antibodies were randomly selected and identified by p H ELISA,from which the positive strains were selected for gene sequencing.3.Use homology modeling and molecular docking technology to evolve single-chain antibodies.First,the sequencing results are compared,and the sequence with the highest homology was selected as the template to establish the single chain antibody homology model and evaluate the model.The ideal model was selected to dock with ENR molecule,and the key force was analyzed,from which the key residues with potential activity were selected for virtual mutation.Then,the optimized mutant was linked with ENR to further verify the feasibility of the model mutation.The single chain antibody was evolved by point mutation technology to obtain the sequence of single chain antibody mutant,and expressed by E.coli Expression System.Finally,protein beads were used to purify.4.Based on Sc Fv mutants,an indirect competitive ELISA method was established.The sensitivity,specificity and accuracy of the method were comprehensively evaluated by establishing standard curve,cross reactivity analysis and recovery experiment.Results1.The complete antigen was identified by UV spectrophotometry and SDS-PAGE.The results showed that the maximum absorption peak and molecular weight of each substance changed before and after coupling.The serum titer of the immunized animals was 1:12800.The total RNA extracted from animal spleen was identified to be complete and of high purity.The length of the heavy chain was about 400 BP,the length of the light chain was about 340 BP,and the length of the single chain antibody was about 780 BP.2.The phage antibody library was constructed with a capacity of 3.45×10⁵.After four rounds of enrichment and screening,the library capacity reached 2.98×10⁷,and the overall enrichment factor was 86.4 times.The positive rate of randomly selected monoclonal phage colonies was 86.7%by Phage-ELISA.The highest positive phage was selected for sequencing,and the corresponding amino acid sequence was obtained.3.After homologous modeling,the model is good and meets the requirements of molecular docking.Molecular docking analysis showed that the hydrophilic carboxyl group of ENR and the carbonyl group of quinoline did not match the hydrophobic residue leu121 of ENR Sc Fv active site.Subsequently,the hydrophobic residue leu121was mutated into hydrophilic residue Asn to obtain the Sc Fv mutant with stronger affinity to ENR.After docking the mutant model with ENR,the binding ability was stronger,and the score decreased from-5.16 to-7.83.The Sc Fv mutant was successfully expressed in E.coli BL21,and the target band of 48 KDa was identified.The Sc Fv mutant was purified by centrifuging the supernatant with 20 mmol/L imidazole.4.Based on Sc Fv mutants,an indirect competitive ELISA method was established.The standard curve was y=-0.2369x+0.7077（R²=0.9725）.The IC₅₀ is 7.53 ng/m L,and the detection range(IC₂₀～IC₈₀)is 0.41～138.99 ng/m L.Compared with the IC₅₀（47.12 ng/m L）of ENR-Sc Fv before mutation,the sensitivity is increased by 6.26 times.The crossover with other antibiotics was less than 0.04%,the average recovery rate on the day of addition was 75.81%～98.08%,and the relative standard deviation was 1.61%～7.45%.The average recovery rate on the third day after addition was 75.54%～97.77%,and the relative standard deviation was 2.87%～10.72%.ConclusionsIn this study,we successfully constructed a large phage antibody library and screened out high specific enrofloxacin Sc Fv.After modification,the established indirect competitive ELISA detection method has high sensitivity,specificity and accuracy.This provides a more accurate and rapid method for the detection of ENR residues in animal food.