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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 chainF v,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27kD)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768bp,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。
In recent years, studies have shown that epidermal growth factor receptor (EGFR) is a very important target in the treatment of cancer. In this study, phage display technology was used to screen EGFR specific single chain F v (scFv). The mice were immunized with A431 high EGFR-expressing human squamous cell carcinoma cells to extract splenocyte mRNA. The VH and VL genes were amplified by RT-PCR and assembled into the scFv gene. The scFv gene was ligated into the phagemid pCANTAB 5E and transformed into E.coli TG1 cells by electroporation to construct a phage scFv library with a capacity of 2.5 × 107. Five cycles of enrichment screening of phage antibody library using purified EGFR as target antigen resulted in secondary antibody library 6F-10. 48 clones were picked for ELISA assay and 45 clones were positive. The clone with the highest positive value was infected with E. coli HB2151 and IPTG induced the expression of scFv. The scFv (about 27 kD) is present in soluble form in the cytoplasm and in the periplasm and can be secreted into the supernatant. Sequencing results showed that the scFv gene was 768 bp in length and encoded 256 amino acids. VH is a heavy chain variable region gene homologous to mouse Ig, and VL is a κ light chain variable region gene. Each of VH and VL consists of three antigen complementarity determining regions and four framework regions. Western blotting and immunofluorescence showed that soluble scFv specifically bound to purified EGFR antigen and to cell surface EGFR, respectively. The acquisition of anti-EGFR specific scFv provides a vector-directed molecule for the development of antibody therapeutics targeting EGFR and research biotherapeutics.