天然兔源噬菌体单链抗体库的构建与鉴定  

Construction and Characterization of Native Rabbit ScFv Phage Display Library

作　　者：黄爱玲[1] 冯荣虎[1] 吕暾[1] 李森[2] 

机构地区：[1]福州大学生物科学与工程学院,福建福州350108 [2]北京师范大学生命科学学院北京市基因工程药物及生物技术重点实验室,北京100875

出　　处：《生物技术》2012年第1期31-34,共4页Biotechnology

基　　金：国家自然科学基金项目("利用大容量噬菌体抗体库筛选有应用价值的抗体型分子伴侣";No.30800188)资助

摘　　要：目的:构建天然兔源噬菌体单链抗体库。方法:采用RT-PCR法从未免疫的兔子脾脏中克隆得到抗体重链可变区(VH)与轻链可变区(VL)基因,重叠PCR将VH和VL拼接成scFv片段,将scFv连接到噬菌粒pComb3XSS上,电转入XL1-Blue菌中,得到单链抗体库,并用此抗体库筛选抗肌酸激酶抗体。结果:构建了容量为4×108,基因重组率95%的单链抗体库,DNA指纹图谱显示抗体库多样性良好。以肌酸激酶为抗原,从该库中筛到3株抗肌酸激酶的抗体。结论:分析表明构建的天然兔源单链抗体库质量良好,可用于快速筛选、制备多种单链抗体。Objective：To construct a native rabbit phage display library of single chain Fv（scFv） antibodies.Method：Rabbit antibody variable heavy（VH） and variable light（VL） genes were amplified by RT-PCR from non-immunized rabbit＇s spleen.Using over-lap PCR obtained the scFv fragments and the products were ligated into the phagemid vector pComb3XSS.The recombinant phagemid was then electroporated into competent E.coli XL1-Blue to construct a rabbit scFv library,then using this library to screen antibodies with affinity for creatine kinase.Result：The results showed that the library contained 4×108 clones,the gene recombination rate was 95% and the library exhibited high diversity by DNA fingerprinting.Three scFv antibodies against creatine kinase were successfully selected from the library by panning.Conclusion：The constructed native rabbit phage scFv library has a high quality and can be used for generating scFv antibodies for antigens.

关 键 词：噬菌体展示 单链抗体 兔源抗体库 

分 类 号：Q785[生物学—分子生物学]