从噬菌体抗体库中淘选血纤维蛋白特异的单链抗体  

Panning of Fibrin-specific Single-chain Antibodies from Phage Antibody Library

作　　者：孙雷[1] 高新胜[1] 黄仪秀[1] 朱圣庚[1] 

机构地区：[1]北京大学生命科学学院,北京100871

出　　处：《中国生物化学与分子生物学报》2000年第6期722-726,共5页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金!资助项目 (396 70 170 ) ;高校博士学科点专项科研基金!资助项目 (95 0 0 12 8)

摘　　要：为构建小鼠噬菌体抗体库 ,以获得对人血纤维蛋白特异的抗体 ,由小鼠脾脏提取 m RNA,经反转录 PCR扩增出抗体重链、轻链可变区基因片段 ,将二者和一段编码十五肽 (Gly4 Ser) 3的 DNA接头借助重组 PCR组装成为单链抗体 (single- chain antibody,Sc Ab)基因 .将单链抗体基因插入噬菌体展示载体 p CANTAB- 5E,通过电击法转化大肠杆菌 TG1细胞 ,用辅助噬菌体 M1 3K0 7超感染 ,构建了库容量在 1 0 8以上的噬菌体单链抗体库 .利用亲和选择方法 (淘选 ) ,从噬菌体抗体库中选得血纤维蛋白特异的单链抗体 .模拟抗体成熟过程 ,用 DNA改组 (DNA shuffling)技术使抗体基因重新组合 ,构建新的改组抗体库 ,并从中选择到提高了亲和力的噬菌体单链抗体 .抗体基因在大肠杆菌中表达 ,表达蛋白经 Sephadex G- 75柱层析分离 ,得到初步纯化的单链抗体蛋白 .A phage antibody library was constructed in order to obtain human fibrin specific antibody.mRNA was isolated from nonimmunized mouse spleens and used as the template for the first strand cDNA synthesis by reverse transcription primed with random hexamers.The variable region cDNA of antibody heavy and light chains (V H and V L) were amplified by polymerase chain reaction (PCR) separately.Both the V H and V L fragments were assembled into a single gene by using a DNA linker encoding a pentadecapeptide(Gly 4Ser) 3 through primary PCR.The assembled single chain antibody (ScFv) DNA fragments were amplified with a set of oligonucleotide primers that introduce restriction sites for cloning via second PCR.The PCR product was digested with restriction enzymes and cloned into the phagemid vector pCANTAB 5E.Then the recombinant DNA was introduced into Escherichia coli TG1 by electroporation.Rescue of recombinant phage antibody library was performed by superinfection of helper phage M13K07. The antibody library contains more than 1×10 8 phage ScFv antibodies.Using affinity selection or panning,the fibrin specific ScFv displayed on the surface of fused phages was isolated from the phage antibody library.Imitating the antibody maturation in vivo, the specific antibody genes were realigned by DNA shuffling,with which a “shuffling library” was constructed after cloning and electroporation.Through panning and screening the secondary library,some specific antibodies with higher activity were selected,whose soluble antibody proteins were expressed in E.coli strain HB2151,and then partially purified by Sephadex G 75 gel filtration chromatography.The fibrin specific ScFv could be useful in constrcting directed thrombolytics or thrombus seeker.

关 键 词：噬菌体抗体库 血纤维蛋白特异抗体 天然抗体 

分 类 号：Q938.48[生物学—微生物学] Q511