Construction And Identification Of Fleroxacin Phage Antibody Libraries

ObjectiveFleroxacin(FLE)is a fluoroquinolone antibacterial drug which is widely used in medical clinical and animal feed additives.Due to its broad-spectrum antibacterial effect,excessive use in animal-derived foods and medicines,it leads to excessive residues.It is a serious threat to human health,and it is necessary to further strengthen monitoring and improve its detection methods.The purpose of this study was to construct a natural murine phage antibody library,from which single-chain antibodies to fleroxacin were screened for expression and identification.Recombinant antibody prepared based on genetic engineering method is the third generation antibody,with short production cycle and easy modification.It provides a new method for the rapid and low-cost preparation of target antibody.Methods1.Non-immunized healthy Balb/c mice were selected,total cellular RNA was extracted,reverse transcribed into cDNA as a template,and the heavy chain variable region and light chain variable region genes of the antibody were amplified by RT-PCR and extended by overlap PCR is linked with Linker(Gly4Ser)3,assembled into single chain antibody genes,and introduced Sfi I and Not I restriction sites.The double-digested target fragment was ligated with the phagemid pCANTAB5E and electrotransformed E.coli TG1 to construct a primary phage antibody library.2.The artificial antigen of fleroxacin was synthesized,FLE-OVA was used as the coating source to conduct three rounds of enrichment screening of the antibody library,and 10 monoclonals were randomly selected for phage-ELISA identification and gene sequencing.3.After fleroxacin single chain antibody and pET-32a vector were digested with Bgl? and Hind ? restriction enzymes,FLE-ScFv was inserted into pET-32a vector with T4 ligase.The E.coli BL21(DE3)strain was transformed and cultured with shaking in LB medium until the OD600 reached 0.7.Then 1.0 mmol/L IPTG was added and placed in a shaker at 20? to induce expression for 10 hours.The bacteria were collected by centrifugation and resuspended in PBS.supernatant and precipitate were collected after ultrasonication.After magnetic beads purification,protein expression was analyzed by SDS-PAGE and Western blot,and the titer and inhibition were identified by ELISA.Results1.The 340bp VH and 320bp VL gene were amplified,and the 750bp single chain antibody fragment was obtained by splice.A primary phage antibody library with the size of 2.1×1011 was successfully constructed.2.The artificial antigen of fleroxacin was successfully synthesized,and the antibody library was subjected to three rounds of enrichment screening with FLE-OVA as the coating source.The results showed that the phage recovery rate increased gradually,and the antibody library was effectively enriched.Phage-ELISA results showed that the single-chain antibody of fleroxacin that could specifically bind to FLE-OVA was successfully screened.3.The results of SDS-PAGE and Western blot of the purified FLE-ScFv showed that the antibodies were mainly expressed as inclusion bodies in E.coli,and the target band of about 47kD was obtained.The results of gene sequencing were analyzed by Ediseq sequence analysis,consistent with the structure and characteristics of single-chain variable region antibody genes.Indirect competitive ELISA results:the linear correlation coefficient of the standard curve regression equation is?0.99,and the IC50 is 42ng/ml,which can detect food and drug residues below the low limit of the national fleroxacin residue standard.ConclusionsIn this study,a library of natural phage single-chain antibodies was successfully constructed,screened and obtained the single-chain antibody that can specifically bind to fleroxacin,providing technical support for the rapid preparation and detection of antibodies and the analysis of food and drug residues.