抗EGFRvⅢ噬菌体抗体库的构建和筛选  

Construction and Screening of Phage Antibody Libraries Against Epidermal Growth Factor Receptor Variant Type Ⅲ

作　　者：周敏[1] 石必枝[1] 顾健人[1] 李宗海[1] 

机构地区：[1]上海交通大学医学院上海市肿瘤研究所癌基因及相关基因国家重点实验室,上海200032

出　　处：《中国生物工程杂志》2010年第4期1-7,共7页China Biotechnology

基　　金：国家"863"计划(2007AA021203);上海市青年科技启明星计划(07QA14046)资助项目

摘　　要：目的:应用噬菌体展示技术筛选针对表皮生长因子受体突变体Ш(epidermal growth factorreceptor variant typeⅢ,EGFRvIII)的单链抗体(single chain Fv,scFv)。方法:利用原核表达纯化的人EGFRvIIIex蛋白和高表达EGFRvIIIex的小鼠成纤维细胞系NIH3T3免疫小鼠,扩增VH和VL片段并拼装成scFv基因,连接至噬菌粒pCANTAB5E,电击转化Hpd3cells,构建噬菌体单链抗体库,并进行3轮富集筛选。在第4轮筛选时,采用了降低抗原浓度的方法。然后将筛选得到的阳性克隆测序分析,转化E.coliHB2151,IPTG诱导可溶性scFv的表达。结果:构建了库容为7.9×107的噬菌体单链抗体库。经过第4轮低浓度抗原筛选,得到了较高亲和力的克隆。取单个阳性克隆测序分析结果表明,该抗EGFRvIII scFv基因序列长807bp,编码268个氨基酸。IPTG诱导后表达的可溶性scFv可分别与纯化的EGFRvⅢex抗原以及细胞表面的EGFRvⅢex结合。结论:利用噬菌体抗体库筛选得到了高亲和力的抗EGFRvⅢ scFv,为开发针对EGFRvⅢ的抗体药物提供了靶向载体分子。Objective：To construct a phage display library of single chain variable fragment （scFv） antibodies and to screen a scFv antibody against epidermal growth factor receptor variant type III （EGFRvIII）.Method：Balb/c mice were immunized by recombinant EGFRvIIIex and NIH3T3/EGFRvIIIex cell line.Genes of variable heavy （VH） and variable light （VL） chains of antibodies were prepared by RT-PCR from the splenic cells of immunized mice and further artificially joined with a flexible peptide linker,then ligated into the phagemid vector pCANTAB 5E.The phagemides containing scFv were transformed into electrocompetent Hpd3cells.The recombinant phages were enriched through 3 rounds of affinity panning and the anti-EGFRvIII phage scFv clones were identified by ELISA.The fourth round of panning performed at a higher stringency of selection,which used a capture concentration of 0.5μg or 0.05μg.After identified by ELISA,one positive clone was sequenced and transfected into E.coli HB2151 cells to express.Result：A phage display library with a complexity of approximately 7.9×107 was constructed.After the fourth round of panning performed at a increasing stringency of selection,higher affinity clones were obtained.Sequencing analysis showed that the anti-EGFRvIII scFv was 807 base pairs coding 268 amino acids.The soluble scFv exhibited the binding activity to purified recombinant EGFRvIIIex protein and EGFRvIII overexpression cell line.Conclusion：The successful preparation of anti-EGFRvIII scFv will provide an EGFRvIII targeted molecule for the development of antibody-based drugs.

关 键 词：表皮生长因子受体突变体Ⅲ 噬菌体抗体库 单链抗体 

分 类 号：Q819[生物学—生物工程]