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

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.