Construction Of Salbutamol Phage Single-chain Antibody Library And Prokaryotic Expression Of Recombinant Antibody Protein

Salbutamol(SAL)is a class of synthetic adrenaline drugs,which is often illegally used as a feed additive in the production of livestock products,posing a huge threat to the safety of animal-derived food in our country.Single-chain antibody(Sc Fv)has gradually become an important research object for establishing rapid immunological detection methods because of its small molecular weight,simple operation and low production cost.Therefore,the purpose of this study is to use phage display technology to construct a mouse-derived immune phage single-chain antibody library,from which to screen out anti-SAL Sc Fv,and provide a theoretical basis and material basis for the later establishment of rapid immunological detection methods.In this experiment,the complete antigen SAL-BSA/OVA was prepared by the mixed acid anhydride method and the activated ester method respectively.After the three immunizations with SAL-BSA,the total RNA was extracted from the mouse spleen,and the Sc Fv gene was obtained by PCR technology and electrotransformed into TG1 competent cells.After infection with helper phage M13K07,the SAL phage single-chain antibody library was constructed.Then,SAL-BSA/OVA was used as the target antigen to carry out four rounds of enrichment and panning of the SAL phage single-chain antibody library,and the identified positive clones were transformed into E.coli HB2151 strain for inducing expression.The osmotic shock method was used to extract the soluble Sc Fv in the periplasmic cavity and its biological activity was identified.The results showed that the complete antigen SAL-BSA/OVA was prepared successfully by SDS-PAGE and ultraviolet spectroscopy scanning method.The antibody titer of mouse serum was about 1:12 800.And the mouse antibody VH,VL and Sc Fv gene fragments were approximately 350 bp,320 bp and 750 bp in size,which were consistent with the expected results.After transformation of the recombinant plasmid Sc Fv-p CANTAB5E whose positive insertion rate was 83.3%,the diversity of the antibody library was 85%,which indicated a phage single-chain antibody library with a storage volume of 5.75×10⁵cfu/m L was obtained.Enrichment panning and sequencing results showed that a SAL-Sc Fv-3 antibody encoding 234amino acids with a size of 702 bps was obtained.The BLAST analysis showed that the VH and VL gene sequences of SAL-Sc Fv-3 have 90.18%and 91.59%homology with mouse immunoglobulin VH and VL?chains,respectively.Ex PASy analysis of the physical and chemical properties of SAL-Sc Fv-3 showed that its molecular weight of SAL-Sc Fv-3 was 25.06 k Da,and its isoelectric point p I and average hydrophilicity was 8.74 and-0.471 respectively.The soluble expression identification result of E.coli HB2151 infection showed that the antibody titer of the periplasmic cavity extract was about 1:320,and the IC₅₀was 294.86?g/L.Except for CLB and CIB,there is no cross-reaction with other structural analogs tested.In summary,this study successfully constructed a SAL murine phage single-chain antibody library with a capacity of 5.75×10⁵cfu/m L,and screened an anti-SAL single-chain antibody with certain biological activity.