| 天然兔源噬菌体单链抗体库的构建与鉴定 |
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| 引用本文： | 黄爱玲,冯荣虎,吕暾,李森.天然兔源噬菌体单链抗体库的构建与鉴定[J].生物技术,2012,22(1):31-34. |
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| 作者姓名： | 黄爱玲 冯荣虎 吕暾 李森 |
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| 作者单位： | 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 |
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| 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。
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| 关 键 词： | 噬菌体展示 单链抗体 兔源抗体库 |
Construction and Characterization of Native Rabbit ScFv Phage Display Library |
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| HUANG Ai-ling
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FENG Rong-hu
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LV Tun
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LI Sen.Construction and Characterization of Native Rabbit ScFv Phage Display Library[J].Biotechnology,2012,22(1):31-34. |
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| Authors: | HUANG Ai-ling FENG Rong-hu LV Tun LI Sen |
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| Institution: | 1.College of Biological Science and Engineering,Fuzhou University,Fuzhou 350108,China; 2.College of Life Sciences,Beijing Normal University,Beijing Key Laboratory of Genetic Engineering Drugs and Biotechnology,Beijing 100875,China) |
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| Abstract: | 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. |
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| Keywords: | phage display single chain Fv rabbit antibody library |
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