半合成抗体库的构建及抗Tie2 Fab抗体的筛选  

Construction of a Semi-synthetic Antibody Library and Screening Anti-Tie2 Fab Antibody

作　　者：廖振林[1] 侯利华[1] 李建民[1] 陈薇[1] 童贻刚[1] 王臣 王慧春[3] 安小平[1] 

机构地区：[1]军事医学科学院流行病与微生物学研究所,北京100071 [2]西北农林大学,杨凌712100 [3]佳木斯大学医学院,佳木斯154007

出　　处：《微生物学通报》2004年第6期37-41,共5页Microbiology China

基　　金：北京市自然科学基金资助项目 (No 5 0 2 2 0 1 2 )

摘　　要：构建一个半合成抗体库 ,不经免疫制备人源抗Tie2Fab抗体。通过RT PCR方法 ,从人脐带血淋巴细胞总RNA扩增轻链基因及重链VH段基因 ,将轻链基因插入pCOMb3载体中 ,得人轻链质粒库 ;从HBsAb的Fd段基因制备含有不同长度随机化CDR3的FR3 CDR3 J CH1片段 ,然后将VH段基因与随机化的CDR3融合 ,得到Fd基因片段 ,再将其插入轻链质粒库中 ,得半合成人Fab质粒库。通过多次建库 ,获得总容量为 2× 1 0 7的半合成抗体库。其Fd段和轻链基因的重组率为 5 0 %。经 3轮淘洗 ,从噬菌体抗体库中筛选到与Tie2抗原特异结合的噬菌体克隆。A semi-synthetic antibody library was constructed,from which anti-Tie2 Fab antibodies were screened .Human immunoglobulin light chain genes and a part of heavy chain variable region genes were amplified by RT-PCR from fetus umbilical cord blood, light chain genes were cloned into pComb3 vector to generate light chain library; FR3-CDR3-J-CH1 segments were amplified from HBsAbs Fd fragment, then VH and synthetic random CDR3 of different length were overlapped by PCR, and inserted into light chain library to form a semi-synthetic phage antibodylibrary. An antibody library containing 2×10 7 cfu was obtained .With more than 50% containing Fd and light chain genes. specific Fab could be selected after three rounds of panning. Then the positive clones were sequenced.

关 键 词：噬菌体抗体库 TIE2 FAB 

分 类 号：R392[医药卫生—免疫学]