Construction Of Murine Phage Antibody Library And Screening Of Anti-Amoxicillin Single Chain Antibodies

Phage antibody library technology is a new antibody preparation technology based on phage display.Because it can obtain high affinity antibodies without artificial immunity and hybridoma technology,it is widely used in food detection,medical testing and other fields.The issue of food safety has always attracted national and public attention.Amoxicillin（AMO）and other penicillin antibiotics are widely used in various veterinary drug preparations,resulting in drug residues in animal derived foods,posing a significant threat to human health.The aim of this study is to construct a large and diverse single-chain variable fragment（sc Fv）antibody library using phage antibody library technology,and screen sc Fv targeting AMO,providing a theoretical basis for the establishment of an immunological rapid detection method for AMO in animal derived foods.This study extracted total RNA from the spleen tissue of healthy BABL/C female mice,synthesized c DNA through reverse transcription,amplified antibody variable region genes（VH,VL）,and spliced them into sc Fv genes through SOE-PCR.The recombinant vector p CANTAB-5E-sc Fv was constructed and electrotransformed into competent cells of E.coli TG1 to obtain a primary antibody gene library with a capacity of 6.5×10⁸cfu/m L.After sequencing identification and sequence analysis,the prepared library showed good diversity.After rescuing the auxiliary bacteriophage M13KO7,a bacteriophage antibody library was obtained with a titer of 5×10¹²pfu/m L。Ovalbumin（OVA）was coupled to AMO by carbodiimide method.The coupling was confirmed to be successful by UV scanning and SDS-polyacrylamide gel electrophoresis（SDS-PAGE）.The concentration of AMO-OVA was 0.6342 mg/m L by Coomassie Brilliant Blue method.Furthermore,using AMO-OVA as the coating material,four rounds of‘adsorption-elution-amplification’biopanning were performed on the phage antibody library to obtain a high affinity phage antibody strain named AMO-sc Fv.After sequencing,the AMO-sc Fv nucleotide sequence was obtained,with a length of 735 bp and encoding 245 amino acids.The VH sequence had 91.2%homology with the mouse derived heavy chain variable region sequence（Sequence ID:BAE71421.1）,and the VL sequence had 92%homology with the mouse derived kappa chain sequence（Sequence ID:AFR53773.1）.The expressed AMO sc Fv has good binding activity to AMO identified by competitive indirect ELISA.Its IC₅₀is 105.9 ng/m L,which has a high cross reaction rate with ampicillin,and a low cross reaction rate with carbenicillin,oxacillin,cloxacillin,penicillin V and cefazolin.The above results indicate the successful construction of a natural mouse derived phage antibody library and the screening of a single chain antibody against AMO,which has a certain sensitivity and specificity.This provides a theoretical basis for the establishment of a rapid immunological detection method for penicillin drugs such as AMO in animal derived foods in the future.