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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 screen single chain Fv (scFv) against epidermal growth factor receptor variant type Ⅲ (EGFRvIII) by phage display technique. Methods: The purified human EGFRvIIIex protein and the mouse fibroblast cell line NIH3T3 with high expression of EGFRvIIIex were used to immunize mice. The VH and VL fragments were amplified and assembled into the scFv gene. The scFv gene was inserted into the phagemid pCANTAB5E and transformed into Hpd3cells by electroporation. Phage single chain antibody library, and three rounds of enrichment screening. At the fourth round of screening, a method of reducing the concentration of antigen was employed. Then the positive clones screened were sequenced and transformed into E.coli HB2151. IPTG induced the expression of soluble scFv. Results: A phage scFv library with a capacity of 7.9 × 107 was constructed. After the fourth round of low concentration antigen screening, higher affinity clones were obtained. Single positive cloning sequencing analysis showed that the anti-EGFRvIII scFv gene sequence is 807bp in length and encodes 268 amino acids. Soluble scFv expressed after induction of IPTG binds to the purified EGFRvIIIex antigen and to the cell surface EGFRvIIIex, respectively. CONCLUSION: High-affinity anti-EGFRvⅢ scFv was screened by using phage antibody library. It provides a target vector for the development of antibody against EGFRvⅢ.