人源性促甲状腺激素受体抗体Fab段抗体库的构建  

Construction of Fab fragment library of human thyrotrophin receptor antibody

作　　者：杜晓明[1] 李宁[2] 宋春青[1] 方佩华[2] 

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,300052

出　　处：《国际内分泌代谢杂志》2015年第4期238-241,共4页International Journal of Endocrinology and Metabolism

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，构建人源性促甲状腺激素受体抗体（TRAh）Fah片段抗体库。方法从甲状腺功能亢进症患者外周血单个核细胞抽提总RNA，PCR法扩增免疫球蛋白分子轻链κ、λ基因及重链Fd基因。构建轻链文库及组合文库。一个随机的组合文库在噬菌体表面表达。结果从外周血单个核细胞中抽提得到总RNA，并反转录获得cDNA文库。PCR法扩增了大小约为680bp的轻链κ、λ基因及重链Fd基因，并构建了库容为1．32×10s基因抗体库（轻链库）和库容为2．28×10 5Fab抗体库（组合文库）。Fah组合文库转化到大肠杆菌XL1-Blue感受态细胞，在辅助噬菌体M13K07的辅助下，扩增得到噬菌体抗体库。结论噬菌体表面展示技术可成功构建人源性TRAbFab片段组合文库。Objective To construct a Fab fragment library of human thyrotrophin receptor antibody （TRAb） using phage display technology. Methods Total RNA was isolated from peripheral blood mononuclear cells of patients with hyperthyroidism. Human immunoglobulin κ/λ light chain and heavy chains Fd genes were amplified by PCR. A light chain library and a combinatorial library were constructed respectively. A random combinatorial library was expressed on the surface of filamentous phage. Results cDNA library was harvested successfully by reverse transcription technology from total RNA of peripheral blood mononuclear cells. Human immunoglobulin 680 bp κ/λ, light chain and heavy chain Fd genes were obtained by PCR. The size of κ light chain library was 1.32 × 105.The actual diversity of Fab combinatorial library was 2.28 × 105.The Fab combinatorial library were transformed in E.coli XL1 Blue.The transformered cells were infected with M13K07 helper phage to amplify phage antibody Fabs. Conclusion A human TRAb Fab fragment phage combinatorial library can be constructed by phage surface display technology.

关 键 词：噬菌体表面展示技术 人源性单克隆抗体 促甲状腺激素受体抗体Fab 

分 类 号：R581.1[医药卫生—内分泌]