甲氧苄啶纳米抗体噬菌体展示文库的构建及初步筛选  

Construction of Phage Display Library and Perliminary Screening of Trimethoprim Nanobody

作　　者：刘翔宇 尹永康 张红 宋茜 王玲[2] 张启迪 梁晓 LIU Xiangyu;YIN Yongkang;ZHANG Hong;SONG Qian;WANG Ling;ZHANG Qidi;LIANG Xiao(College of Veterinary Medicine,Qingdao Agricultural University,Qingdao 266109,China;Qingdao Agricultural University Hospital,Qingdao 266109,China)

机构地区：[1]青岛农业大学动物医学院,青岛266109 [2]青岛农业大学校医院,青岛266109

出　　处：《中国畜牧兽医》2024年第11期5074-5085,共12页China Animal Husbandry & Veterinary Medicine

基　　金：山东省自然基金面上项目(ZR2020MC187);国家大学生创新项目(202210435028);青岛市科技惠民示范专项项目(24-1-8-xdny-21-nsh、24-1-8-xdny-29-nsh)。

摘　　要：【目的】构建甲氧苄啶(trimethoprim,TMP)纳米抗体(nanobody,Nb)噬菌体展示文库,制备TMP高亲和力纳米抗体,为TMP免疫分析方法的建立提供新型抗体材料。【方法】通过活性酯酶法将TMP半抗原Hapten B与牛血清白蛋白(BSA)、钥孔血蓝蛋白(KLH)偶联制备2种免疫原,Hapten A和Hapten B与卵清白蛋白(OVA)偶联制备2种包被原。采用磺胺增效剂类药物单克隆抗体5C4,通过间接ELISA方法验证免疫原和包被原偶联效果。将免疫原与弗氏佐剂混合,先后对羊驼进行基础免疫和加强免疫,监测每次免疫后羊驼抗血清效价及对TMP的亲和力,选取效价及TMP亲和力最高的外周血作为纳米抗体基因来源,构建噬菌体展示文库。分离羊驼外周血总淋巴细胞,扩增重链抗体可变区纳米抗体基因(VHH),插入噬菌粒,电击转化大肠杆菌XL1-Blue感受态细胞构建初始噬菌体纳米抗体展示文库。经辅助噬菌体救援,采用同源包被原浓度梯度递减包被、酸洗脱、TMP浓度梯度递减洗脱筛选策略筛选TMP高亲和力噬菌体并测序,原核表达纳米抗体,通过SDS-PAGE和Western blotting鉴定纳米抗体融合蛋白。【结果】间接ELISA检测结果显示,2种免疫原与2种包被原在稀释18000倍时D 450 nm值仍高于1.25。免疫原Hapten B-BSA 5次免疫后羊驼抗血清效价为1∶88000,对TMP的半数抑制浓度(IC 50)为12.21μg/L,但Hapten B-KLH 4次免疫后羊驼抗血清效价为1∶11000,对TMP的IC 50为30.56μg/L。以Hapten B-BSA五免后羊驼外周血为纳米抗体基因来源构建噬菌体展示文库,初始库容量为6.08×10^(7) CFU/mL,目的片段插入率为96%,噬菌体展示文库多样性良好。经4轮筛选后,获得3株TMP高亲和力噬菌体,测序分析后获得1株TMP高亲和力纳米抗体及序列。SDS-PAGE和Western blotting鉴定融合蛋白大小约为17 ku,蛋白含量80%以上。【结论】本研究成功构建TMP纳米抗体噬菌体展示文库,获得1株TMP高亲和力纳米抗【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 18000 times.Following five immunizations with Hapten B-BSA,antiserum titers reached 1∶88000 with an half maximal inhibitory concentration(IC 50)of 12.21μg/L for TMP.And antiserum titers reached 1∶11000 with an IC 50 of 30.56μg/L for TMP following

关 键 词：甲氧苄啶 纳米抗体 噬菌体展示文库 

分 类 号：S852.43[农业科学—基础兽医学]