Abstract: 【Objective】 This study was aimed to construct an trimethoprim (TMP) nanobody phage display library and prepare a high-affinity TMP nanobody,provide novel antibody materials for the establishment of TMP immunoassay methods. 【Method】 Two immunogens were prepared by conjugating Hapten B to bovine albumin (BSA) and keyhole hemocyanin (KLH) by the active ester method.And Hapten A and Hapten B were conjugated to ovalbumin (OVA) to prepare two coating antigens using the same method.The conjugating effect was identified by indirect ELISA with the monoclonal antibody 5C4 against sulfonamide synergists.The immunogens were mixed with Freund’s adjuvants for primary and booster immunizations of alpacas,antiserum titers and TMP affinity were monitored after each immunization.Peripheral blood samples with the highest titer and maximal affinity for TMP was selected as the source of nanobody genes to construct the phage display library.Alpaca peripheral blood lymphocytes were isolated,variable domain of heavy-chain antibody (VHH) gene was amplified by PCR,and then ligated into phagemids.The recombinant phagemids were transformed into E.coli XL1-Blue competent cells to construct the initial phage display library,and then rescued with the helper phage.High-affinity TMP phages were obtained by screening using the decreasing concentration gradient coating of homologous coating antigen,acid elution and the decreasing concentration gradient elution of TMP.The sequence of the high-affinity TMP nanobody was obtained by sequencing.The nanobody fusion protein was expressed in prokaryotes and identified by SDS-PAGE and Western blotting. 【Result】 The results of indirect ELISA showed that the D_{450 nm} values of two immunogens and two coating antigens were higher than 1.25 when they were diluted 18 000 times.Following five immunizations with Hapten B-BSA,antiserum titers reached 1∶88 000 with an half maximal inhibitory concentration (IC₅₀) of 12.21 μg/L for TMP.And antiserum titers reached 1∶11 000 with an IC₅₀ of 30.56 μg/L for TMP following four immunizations with Hapten B-KLH.The peripheral blood of alpaca immuned with Hapten B-BSA for five times was selected as the source of VHH and the phage display library was constructed,which exhibited a capacity of 6.08×10⁷ CFU/mL,a 96% insertion rate of target fragment and considerable diversity.After 4 rounds of screening,three high-affinity TMP phages were obtained and 1 sequence of high-affinity TMP nanobody was obtained by sequencing.The size of fusion protein was approximately 17 ku,with more than 80% protein content determined by SDS-PAGE and Western blotting analysis. 【Conclusion】 The TMP nanobody phage display library was successfully constructed,and a high-affinity TMP nanobody was selected from this library.This study provided theoretical basis and technical support for the preparation of nanobodies against sulfonamide synergists,and offered new insights for the preparation of novel recognition materials for veterinary drugs and other small molecules.

Key words: trimethoprim; nanobody; phage display library