目的： 制备靶向表皮生长因子受体（epithelial growth factor receptor，EGFR）隐蔽表位（287-302）的免疫毒素，并鉴定其生物学功能。 方法： 通过基因工程方法将抗EGFR（287-302）的806单链抗体（806 single-chain antibody fragment， 806scFv ）基因经柔性肽与铜绿假单胞菌外毒素A（Pseudomonas exotoxin A， PEA）的截短形式PE38KDEL连接，构建原核表达载体pET-22b-806scFv-PE38KDEL并转化至大肠杆菌BL21（DE3）中，经纯化获得该免疫毒素融合蛋白806scFv-PE38KDEL，ELISA和流式细胞术检测其与EGFR的结合活性，间接免疫荧光检测重组免疫毒素的内化作用，CCK-8法检测806scFv-PE38KDEL对人脑胶质瘤细胞U87MG和U87MG-EGFRvⅢ、表皮癌细胞A431、乳腺癌细胞MDA-MB-468、舌癌细胞CAL-27的细胞毒性。 结果： 成功构建重组免疫毒素806scFv-PE38KDEL，诱导表达的蛋白806scFv-PE38KDEL以包涵体形式存在，经纯化后的纯度>95%，经SDS-PAGE和Western blotting 鉴定为目的蛋白。806scFv-PE38KDEL 能EGFRvⅢ胞外段蛋白结合，还能与外源性表达EGFRvⅢ的肿瘤细胞和高表达EGFR的肿瘤细胞相结合，而与表达低水平EGFR的肿瘤细胞不结合。806scFv介导了重组免疫毒素的内化。806scFv-PE38KDEL对靶细胞有明显的杀伤作用，对过表达EGFRvⅢ的U87MG-EGFRvⅢ细胞IC50值为（5.85±0.03） ng/ml，对EGFR高表达细胞MDA-MB-468、A431、CAL-27的IC50值分别为（162.80±0.06）、（75.72±0.04）、（123.70±0.03） ng/ml。在1 μg/ml的质量浓度下，相比PBS对照组，806scFv-PE38KDEL对U87MG-EGFRvⅢ 、MDA-MB-468、A431和CAL-27细胞增殖的抑制率均显著增高\[（98.67±0.07）% vs （2.45±2.85）%、（86.26±101）% vs （0.48±1.76）%、（96.72±016）% vs （1.33±1.31）%、（96.29±0.30）% vs （2.00±0.60）%，均P<0.01\]，而对U87MG细胞几乎没有抑制作用\[（359±2.09）% vs （0.19±0.95），P>0.05\]。 结论： 本研究所制备的靶向EGFR（287-302）表位的重组免疫毒素806scFv-PE38KDEL能特异地结合并杀伤EGFRvⅢ或EGFR高表达的肿瘤细胞。

Objective: To prepare recombinant immunotoxin targetting a cryptic epitope (287-302 residues) in epithelial growth factor receptor (EGFR) and to explore its biological properties. Methods: Prokaryotic expression vector pET-22b-806scFv-PE38KDEL encoding anti-EGFR (287-302) 806 single-chain antibody (806scFv) fused with PE38KDEL, a truncated form of pseudomonas exotoxin A (PEA), via a flexible peptide was constructed. The immunotoxin fusion protein (806scFv-PE38KDEL) was expressed in E. coli strain BL21 (DE3) and purified. The binding capacity of the immunotoxin to EGFR was detected by ELISA and flow cytometry. The internalization of immunotoxin was showed via indirect immuno-fluorescent assay. The cytotoxicity effect of the immunotoxin on human glioma cell U87MG and U87MG-EGFRvⅢ, epidermis tumor cell A431, breast cancer cell MDA-MB-468 and tongue cancer CAL-27 cell lines were assessed by CCK-8 assay. Results: The recombinant immunotoxin 806scFv-PE38KDEL was constructed successfully. The induced expression product 806scFv-PE38KDEL existed in a form of inclusion body and the purity was above 95% after purification. The protein was identified by SDS-PAGE and Western blotting. 806scFv-PE38KDEL can bind to the protein of EGFRvⅢ extracellular domain and also the cancer cells with exogenous EGFRvⅢ or with a endogenous EGFR overexpression but not to the cancer cells with a low level of endogenous EGFR. The immuno-fluorescent assay showed that the internalization of immunotoxin was mediated by 806scFv. 806scFv-PE38KDEL showed the cytotocicity on targeted cells with EGFRvⅢ overexpression such as U87MG-EGFRvⅢ cells with IC50 values of (5.85±0.03) ng/ml (P<0.01) and EGFR overexpression cancer cells such as MDA-MB-468, A431 and CAL-27 cells with IC50 values of (162.80±0.06) ng/ml, (75.72±0.04) ng/ml, (123.70±0.03) ng/ml, respectively. 806scFv-PE38KDEL almost completely inhibited the growth of cancer cells such as U87MG-EGFRvⅢ, MDA-MB-468, A431 and CAL-27 cells compared the control group, and the inhibitory rates significantly increased (\[98.67±0.07\]% vs \[2.45±2.85\]%, \[8626±1.01\]% vs \[0.48±1.76\]%, \[96.72±0.16\]% vs \[1.33±1.31\]%, \[96.29±0.30\]% vs \[2.00±060\]%; P<0.01) but no effect on U87MG cells (\[3.59±2.09\]% vs \[0.19±0.95\], P>005). Conclusion: Recombinant immunotoxin 806scFv-PE38KDEL that targeted to EGFR (287-302) epitope prepared in this study is a candidate cancer therapeutic agent which can selectively bind and significantly inhibit the growth of the cancer cells with EGFRvⅢ or EGFR overexpression.

上海市科委生物医药重点科技攻关项目资助（No. 10431903700）; “十二五”国家重大新药创制专项资助（No. 2012ZX09103301-005）