人源性TRAb Fab段单克隆抗体的筛选和鉴定  

Selection and identification of human monoantibody TRAb Fab fragment

作　　者：杜晓明[1] 李宁[2] 方佩华[2] DU Xiao-ming;LI Ning;FANG Pei-hua(Department of Endocrinology, Tianjin 4th Center Hospital, Tianjin 300140, China;Department of Nuclear Medicine, Tianjin Medical University General Hospital)

机构地区：[1]天津市第四中心医院内分泌科,300140 [2]天津医科大学总医院核医学科

出　　处：《天津医药》2017年第8期851-855,共5页Tianjin Medical Journal

基　　金：天津市卫生局科技重点项目(2013KR04)

摘　　要：目的通过噬菌体表面展示技术,运用已构建的人源性噬菌体抗体库,筛选、表达人源性促甲状腺激素受体抗体(TRAb)Fab段。方法以人TSHR膜外区的氨基端(h TSHRn)、人TSHR膜外区的羧基端(hTSHRc)的融合蛋白为抗原,通过数轮"吸附-洗脱-扩增"富集噬菌体抗体,筛选阳性克隆。从已构建的抗体库中筛选到甲状腺刺激性抗体(TSAb)Fab、甲状腺阻断性抗体(TBAb)Fab,噬菌体(Phage)-ELISA检测选取阳性克隆,PCR及双酶切鉴定TRAb阳性克隆。检测可溶性TRAb Fab片段阳性克隆的表达,Western blotting鉴定TRAb阳性克隆的免疫学活性,对TRAb Fab阳性克隆进行测序分析。结果经过5轮富集筛选,成功筛选到特异性TRAb(TSAb、TBAb)Fab噬菌体抗体,产率分别提高约77、94倍。通过Phage-ELISA检测,鉴定出了具有抗原结合活性的单克隆抗体,实现可溶性表达。Western blotting证实其免疫学活性。测序分析单克隆48的轻链可变区与人的免疫球蛋白轻链Vλ同源性达到94.4%,重链可变区与人的免疫球蛋白IgG重链VH4同源性为88.9%。克隆56的轻链可变区与人的免疫球蛋白轻链Vλ同源性达到95.6%,重链可变区与人的免疫球蛋白IgG重链VH3同源性为84.6%。结论本研究通过成功筛选获得人源性TRAb(TSAb、TBAb)Fab片段的单克隆抗体。Objective To select and express a human thyrotrophin receptor antibody(TRAb)Fab fragment from phageantibody library constructed with phage display technology.Methods With immobilized antigen,the reconstructedhumanized TRAb Fab library was enriched by five rounds panning(adsorption-elution-amplification).The TSAb Fab andTBAb Fab fragment were selected by coated fusion proteins of hTSHRn and hTSHRc.The positive clones were identifiedand selected by Phage-ELISA.TRAb positive clones were identified by PCR and double restriction enzyme digestion.Thesoluble TRAb(TSAb,TBAb)Fab fragments were expressed.TRAb(TSAb,TBAb)Fab fragments were identified by Westernblotting assay.The DNA fragment was sequenced from the positive clones.Results Following five rounds of biopanning,TRAb(TSAb,TBAb)Fab phage antibody was screened.The enrichment effect reached to77times and94times.The solubleTRAb(TSAb,TBAb)Fab antibodies were expressed from positive clones and identified by phage ELISA.Western blottinganalysis showed that the phage displaying Fab had significant binding activity with antigens.These sequence analysisshowed that all of the heavy chain Fd gene and light chain gene were derived from human immunoglobulin variable region.The light chain variable region of the monoclonal48was homologous to the immunoglobulin light chain Vλhomology of94.4%,and the heavy chain variable region of the monoclonal48was homologous to the immunoglobulin heavy Fd chainVH4homology of88.9%.The light chain variable region of the monoclonal56was homologous to the immunoglobulin lightchain Vλhomology of95.6%,and the heavy chain variable region of the monoclonal56was homologous to theimmunoglobulin heavy Fd chain VH3homology of84.6%.Conclusion We have successfully selected TRAb(TSAb,TBAb)Fab fragment from a human phage display immune antibody library.

关 键 词：受体 促甲状腺素 抗体 单克隆 免疫球蛋白Fab片段 细菌噬菌体 噬菌体展示库 

分 类 号：R581[医药卫生—内分泌]