生长抑素噬菌体展示鼠源单链抗体库的构建与鉴定  

Construction of a Phage Display Library of Repertoire Single Chain Antibody from Mouse Immunized against Somatostatin and Its Identification

作　　者：张颖[1] 侯化鹏[2] 王海瑞[1] 白莉雅[1] 杨利国[1] 

机构地区：[1]华中农业大学动物科技学院动物遗传育种与繁殖教育部重点实验室,武汉430070 [2]华中农业大学动物医学院,武汉430070

出　　处：《农业生物技术学报》2009年第3期375-380,共6页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金项目(No.30771549);国家支撑计划项目(No.2006BAD14B08-02)资助

摘　　要：应用噬菌体表面展示技术,从生长抑素免疫的BALB/C小鼠(mus musculus)脾脏中提取总RNA,用RT-PCR技术反转录成cDNA,通过PCR扩增出抗体重链可变区VH基因和轻链可变区VL基因,再用编码(G1y4Ser)3的Linker在体外将VH和VL连接成单链抗体(ScFv)基因,克隆到噬菌粒载体pCANTAB5E中,电转化至感受态的大肠杆菌(Escherichia coli)TG1,经辅助噬菌体M13K07超感染,形成噬菌体单链抗体库。经检测,噬菌体的滴度为3.5×1011pfu,有效库容为9.3×107。随机挑取20个克隆,PCR方法测定文库重组率为85%,小规模选取阳性克隆测序分析表明,均含有ScFv基因的完整序列,基本满足建库的要求。A library of repertoire immunoglobulin from mouse （Mus musculus） immunized against somatostatin was constructed by a phage display technique. The heavy-chain and light-chain variable region genes （VH and VL） repertoire of immunoglobulin were amplified respectively from the spleen cell RNA by both RT-PCR and a DNA linker encoding （Gly4Ser）3 as a single chain （ScFv） DNA fragment with overlap extension. These fragments were cloned into the phagemid pCANTAB5E and the recombinant phagemid was transformed to susceptible Escherichia coli TG1 by electroporation. In the presence of helper phage M13KO7, the ScFv fusion protein was displayed on the surface of recombinant phages. The titer of the phage antibody library was about 3.5 × 10^11 pfu and contained 9.3 ×10^7 independent clones. Sequencing and bioinformatics analysis of 20 random picked clones showed that the recombinant plasmids contained an intact ScFv gene with a recombination rate of 85%. These results indicated that the normalized phage antibody library was constructed successfully.

关 键 词：生长抑素 噬菌体展示 单链抗体 

分 类 号：S188[农业科学—农业基础科学]