靶向表皮生长因子受体的寡肽与力达霉素强化融合蛋白的构建及其抗肿瘤活性

背景与目的：表皮生长因子受体（epidermal growth factor receptor，EGFR）在多种肿瘤细胞表面异常表达。力达霉素是从链霉菌中分离得到的具有强抗肿瘤作用的烯二炔类抗生素。本研究的目的是制备一种特异性靶向EGFR的寡肽配体与力达霉素的强化融合蛋白（Ec—LDP-AE），并初步探讨其抗肿瘤活性。方法：制备融合蛋白（Ec-LDP），并分离纯化，用高效液相色谱法检测Ec-LDP的纯度，ELISA、流式细胞技术以及细胞免疫荧光方法分析纯化的Ec-LDP蛋白对不同肿瘤细胞的免疫结合活性。将纯化的Ec-LDP蛋白与力达霉素发色团进行组装，构建Ec-LDP-AE，用高效液相色谱法检测350nm处的吸收峰。MTY法检测Ec—LDP-AE的体外抗肿瘤活性。结果：成功构建并表达了Ec-LDP蛋白，目的蛋白分泌至大肠杆菌周质腔中，产量为每升发酵液18mg活性蛋白，其纯度为95．3％。ELISA和细胞流式检测结果均显示Ec-LDP蛋白对EGFR高表达的肿瘤细胞系A431和MCF-7都有很强的免疫结合活性．而对不表达EGFR的NIH3T3细胞则无结合活性。免疫荧光实验也证实Ec—LDP蛋白可与A431细胞膜受体结合。Ec-LDP-AE蛋白在350nm波长处出现特定吸收峰，表明其构建成功。MTT法检测结果显示Ee-LDP—AE对体外培养的肿瘤细胞有强烈的杀伤作用，对MCF-7和A431细胞的IC。值分别为3．06×10^11mol/L和9．38×10^-13mol／L。结论：本研究制备的强化融合蛋白Ec—LDP—AE对表皮生长因子受体有靶向作用，并且保留了力达霉素的强烈细胞毒作用。

Construction of energized fusion protein consisting of epidermal growth factor receptor oligopeptide ligand and lidamycin and its antitumor activity

Background and Objective. Epidermal growth factor receptor （EGFR） is abnormally overexpressed on many kinds of tumor cells. Lidamycin is an enediyne antibiotic with highly potent antitumor activity. This study was to construct a novel fusion protein by recombining EGFR specific oligopeptide ligand and lidamycin, and investigate its antitumor efficacy. Methods：The fusion protein （Ec-LDP） was expressed in E.cofi and purified by affinity chromatography. The purity of Ec-LDP was analyzed by high performance liquid chromatography （HPLC）. ELISA, flow cytometry （FCM） and immunofluorescence assay were used to analyze the binding activity of Ec-LDP to different cancer cell lines. The energized fusion protein Ec-LDP-AE was prepared by integrating the active enediyne chromophore （AE） of lidamycin into the Ec-LDP protein. The cytotoxicity of Ec-LDP-AE was measured by MTT assay. Results： Ec-LDP fusion protein was successfully constructed and secretorily expressed in E.coli, and the production of Ec- LDP protein was 18 mg per liter fermentation broth. The purity of Ec-LDP protein was 95.3%. Ec-LDP protein had strong binding activity to cancer cell lines highly expressing EGFR, such as MCF-7 and A431 cells. However, Ec- LDP had no binding activity to EGFR negative NIH 3T3 cells. The energized fusion protein Ec-LDP-AE showed potent cytotoxicity to MCF-7 and A431 cells with the half maximal inhibitory concentration （ICso） values of 3.06×10^-11 mol/L and 9.38×10^-13 mol/L, respectively. Conclusion. The energized fusion protein Ec-LDP-AE binds to EGFR specifically and kills cancer cells efficiently.