抗表皮生长因子受体噬菌体抗体库的构建筛选及单链抗体可溶性表达

近年来的研究表明，表皮生长因子受体 (epidermal growth factor receptor, EGFR) 是肿瘤治疗中一个很重要的靶点。本研究应用噬菌体展示技术筛选EGFR特异性单链抗体 (single chain Fv, scFv)。利用高表达EGFR的人鳞状上皮癌细胞A431免疫小鼠，提取脾细胞mRNA，RT-PCR扩增VH和VL基因并拼装成scFv基因。将scFv基因连接到噬菌粒pCANTAB 5E中，电击转化E.coli TG1细胞，构建了库容为2.5×10⁷的噬菌体单链抗体库。用纯化的EGFR为靶抗原对噬菌体抗体库进行5轮富集筛选，得到次级抗体库6F-10。挑取48个克隆进行ELISA测定，45个克隆为阳性。取阳性值最高的克隆感染E.coli HB2151，IPTG诱导scFv的表达。scFv (约27 kD) 以可溶形式存在于细胞质及细胞周质中，并可分泌至上清液。测序结果表明，scFv基因序列全长768 bp，编码256个氨基酸。VH为与小鼠Ig同源的重链可变区基因，VL为κ型轻链可变区基因；VH和VL均由3个抗原互补决定区和4个框架区构成。免疫印迹和细胞免疫荧光显示，可溶性scFv可分别与纯化的EGFR抗原以及细胞表面的EGFR发生特异性结合。抗EGFR特异性scFv的获得，为研制靶向EGFR的抗体药物与研究生物治疗提供导向载体分子。

Construction and screening of phage antibody libraries against epidermal growth factor receptor and soluble expression of single chain Fv

Recent studies have shown that epidermal growth factor receptor (EGFR) is an important target for cancer therapy.  The present study prepared single chain Fv (scFv) directed against EGFR.  Balb/c mice were immunized by human carcinoma A431 cells, and total RNA of the splenic cells was extracted.  VH and VL gene fragments were amplified by RT-PCR and further joined into scFv gene with a linker, then scFv gene  fragments were ligated into the phagemid vector pCANTAB 5E.  The phagemides containing scFv were   transformed into electro-competent E.coli TG1 cells.  The recombinant phage antibody library was constructed through rescuing the transformed cells with help phage M13K07.  The specified recombinant phages were   enriched through 5 rounds of affinity panning and the anti-EGFR phage scFv clones were screened and identified with ELISA.  A total of 48 clones from the library were selected randomly and 45 clones were identified   positive.  After infecting E.coli HB2151 cells with one positive clone, soluble recombinant antibodies about  27 kD were produced and located in the periplasm and the supernatant.  The result of sequencing showed that the scFv gene was 768 bp, which encoded 256 amino acid residues.  VH and VL including 3 CDRs and 4 FRs, respectively, were all homologous to mouse Ig.  The soluble scFv showed the specific binding activity to purified EGFR and EGFR located in carcinoma cell membrane.  The successful preparation of anti-EGFR scFv will  provide an EGFR targeted molecule for the development of antibody-based drugs and biological therapy of cancer.