人源性TRAbFab段单克隆抗体的筛选和鉴定

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（hTSHRn）、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TRAbFab片段阳性克隆的表达，Westernblotting鉴定TRAb阳性克隆的免疫学活性，对TRAbFab阳性克隆进行...

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Bibliographic Details
Published in天津医药 Vol. 45; no. 8; pp. 851 - 855
Main Author 杜晓明;李宁;方佩华
Format Journal Article
LanguageChinese
Published 2017
Subjects
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
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ISSN0253-9896

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Summary: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（hTSHRn）、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TRAbFab片段阳性克隆的表达，Westernblotting鉴定TRAb阳性克隆的免疫学活性，对TRAbFab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。Westernblotting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。
Bibliography:12-1116/R
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 t
ISSN:0253-9896