Construction Of Danofloxacin Phage Single-Chain Antibody Library And Screening,Expression,Identification Of The Specific Single-Chain Antibody

Danofloxacin（DAN）has a broad antibacterial spectrum and strong bactericidal effect.It is often used as a feed additive to prevent livestock and poultry diseases.However,massive abuse about DAN has caused a large number of drug residues in livestock products,which seriously endangers human health.In order to regulate the use of DAN and ensure the safety of animal-derived food and human health,this study screened the specific single-chain variable fragment（sc Fv）of DAN by constructing the DAN phage antibody library,which laid the foundation for the evolution of antibody in vitro and the establishment of immunological detection method for DAN residue.In this reaserch,complete antigen DAN-BSA and DAN-OVA were prepared by mixed anhydride method.After identification by SDS-PAGE and UV spectrum scanning,BALB/c mice were immunized with DAN-BSA,and the titer was determined after 4 immunization.The variable domain of heavy chain（VH）,variable domain of light chain（VL）and sc Fv genes’s PCR reaction template was c DNA which extracted RNA from mouse spleen and reversely transcribed.The recombinant plasmid p CANTAB5E-sc Fv was constructed and transformed into E.coli TG1.The transformants were infected with the auxiliary phage M13K07 to construct the single-chain antibody library of DAN.The phage library was alternately enriched by five rounds of affinity selection using DAN-BSA and DAN-OVA as target antigens,so that the anti-DAN phages were enriched.The single-chain antibody sc Fv with high affinity was screened by phage-ELISA after selection,and ligated with prokaryotic expression vector p ET-22b to construct the recombinant plasmid p ET22b-sc Fv.The recombinant plasmid was transformed into E.coli DE3 for protein induction and expression,in order to indentified the immunological activity of the recombinant antibody.The results showed that the complete antigens DAN-BSA and DAN-OVA were successfully prepared,and the serum antibody titer reached 1:25600 after DAN-BSA immunized mice.VH,VL and sc Fv genes were successfully amplified,and the sizes were about 350 bp,320 bp and 750 bp,respectively.After the sc Fv was cloned into p CANTAB5E vector,the DAN phage single-chain antibody library was successfully constructed.The positive insertion rate of the DAN phage antibody library was91.6%,the diversity was 86.4%,the library volume was 3.7×10⁵ pfu/m L,and the titer was 8.1×10¹² pfu/m L.The anti-DAN antibody sc Fv-9 was screened from the antibody library with target antigen DAN-BSA and DAN-OVA.The sc Fv-9 was expressed as inclusion body in E.coli.After dialysis and renaturation,the IC₅₀ of the antibody protein was 149.10 ng/m L,the lowest detection limit was 23.62 ng/m L,and no cross-reactivity rate with CIP,NOR,OFL and SAR.In this study,the anti-DAN single-chain antibody wsa successfully screened from the constructed DAN phage antibody library,which laid the foundation for the later in vitro evolution of antibodies and the establishment of rapid detection methods for DAN immunity.