从大容量噬菌体抗体库中筛选抗DNA-PKcs人源单链抗体  

Screening of Human Anti-DNA-PKcs scFv from Large Scale Phage Library

作　　者：杜丽[1] 周丽君[2] 潘秀颉[1] 王豫[1] 王欲晓[2] 杨陟华[1] 徐勤枝[1] 张士猛[1] 朱茂祥[1] 周平坤[1] 

机构地区：[1]军事医学科学院放射与辐射医学研究所,北京100850 [2]中国人民解放军海军总医院中心实验室,北京100037

出　　处：《中国生物工程杂志》2008年第9期20-26,共7页China Biotechnology

基　　金：国家“973”计划(2007CB914603);国家自然科学基金(30672429);北京市自然科学基金(7072057)资助项目

摘　　要：目的:从单链大容量噬菌体抗体库中筛选特异性的抗DNA-PKcs的人源抗体,用于肿瘤治疗或诊断目的。方法:经抗原性分析及BLAST比对,选定人DNA-PKcs蛋白中抗原性高且与其他蛋白没有同源性的片段,进行原核表达及纯化后将其固定在抗原管上,通过4轮"吸附-洗脱-扩增"过程从大容量抗体库中筛选特异性抗体,转化HB2151菌,制备抗DNA-PKcs的可溶性单链抗体;ELISA检测抗原-抗体结合活性。结果:经生物信息学分析,确定抗原性高且与其他蛋白没有同源性的DNA-PKcs片段DPK3(250个AA)、DPK4(257个AA)。经过4轮筛选,获得26个特异性结合DPK3及31个特异结合DPK4的克隆,指纹分析分别有5种和21种不同的可变区片段;成功制备了可溶性抗体。并做了抗原结合活性鉴定。结论:利用单链大容量抗体库获得抗DNA-PKcs的噬菌体抗体基因并且成功制备成可溶性抗体,为今后的研究和应用奠定了基础。Objective： To clone the genes encoding human anti-DNA-PKcs antibodies from large phage antibody library for the use of cancer therapy or diagnosis in future. Method： Human DNA-PKcs protein fragments with high antigenicity and low homologous to other proteins were defined through antigenicity analysis and BLAST searching. After prokaryotic expression and purification, the protein segments were coated onto immuno-tube. Panning of large scale phage library against antigen was conducted to select specific antibodies against DNA-PKcs. The binding activity and specificity were tested by ELISA method. Soluble scFv was prepared through transfecting HB2151 with the selected phage antibody genes. Result： 250 AA of DPK3 and 257 AA of DPK4 segments were defined as high antigenicity and low homology to other protein through bioinformatic analysis. After 4 rounds of panning, 26 clones were identified with the specific binding ability with DPK3, and 31 clones with DPK4. DNA fingerprinting revealed 5 individual positive clones to DPK3 and 21 individual positive clones to DPK4. Sequencing analysis showed that variable regions of these ScFvs belong to different subgroups. Conclusion： Human DNA-PKcs antibody genes were successfully obtained from large scale phage antibody library.

关 键 词：大容量噬菌体抗体库 DNA依赖蛋白激酶催化亚单位 单链抗体 

分 类 号：Q511[生物学—生物化学]