以人表皮生长因子受体2为靶点的强化融合蛋白LDP-Hr-AE的构建及抗肿瘤活性研究  

Construction of an Energized Fusion Protein LDP-Hr-AE Targeting Human Epidermal Growth Factor Receptor 2 and Its Antitumor Activity

作　　者：郭晓芳[1] 朱小飞[1] 钟根深[2] 甄永苏 

机构地区：[1]新乡医学院,河南新乡453003 [2]新乡医学院第一附属医院,河南新乡453100 [3]中国医学科学院-北京协和医学院医药生物技术研究所,北京100050

出　　处：《中国药学杂志》2012年第12期941-947,共7页Chinese Pharmaceutical Journal

摘　　要：目的制备一种以人表皮生长因子受体2(human epidermal growth factor receptor 2,HER2)为靶点的由抗体重链超变区多肽与力达霉素组成的强化融合蛋白LDP-Hr-AE,并初步探讨其抗肿瘤活性。方法通过聚合酶链式反应扩增出抗人表皮生长因子受体2抗体C6.5重链CDR3区的20个氨基酸残基基因,将其与力达霉素辅基蛋白基因连接构建出融合蛋白基因ldp-Hr,转化至大肠杆菌中进行诱导表达。融合蛋白LDP-Hr采用HisTrap Ni2+亲和色谱柱进行分离纯化,高效液相色谱法检测其纯度。细胞免疫荧光法和基于流式细胞术的亲和实验分析融合蛋白与肿瘤细胞的结合活性。纯化的LDP-Hr蛋白与力达霉素发色团在体外进行组装,构建出强化融合蛋白LDP-Hr-AE。采用MTT法检测强化融合蛋白对肿瘤细胞的杀伤活性,Annex-in V-FITC/PI双染结合流式细胞术的方法检测LDP-Hr-AE蛋白对细胞凋亡的影响。结果成功构建并表达了融合蛋白LDP-Hr,目的蛋白以可溶形式分泌至大肠杆菌培养液和周质腔中,产量可达每升发酵液40 mg活性蛋白,纯化后的蛋白经高效液相色谱法检测纯度为97.4%。细胞免疫荧光实验和基于流式细胞术的亲和实验检测结果均显示LDP-Hr蛋白与HER2高表达的肿瘤细胞系(如SK-BR-3和SK-OV-3细胞)都有很强的结合活性。LDP-Hr蛋白与力达霉素发色团在体外组装后经高效液相色谱法检测350 nm处出现特定吸收峰,表明强化融合蛋白LDP-Hr-AE构建成功。采用MTT法检测了强化融合蛋白的体外杀伤活性,结果显示,LDP-Hr-AE对肿瘤细胞有强烈的杀伤作用,且其杀伤活性要高于力达霉素。细胞凋亡检测结果也表明LDP-Hr-AE在极低的浓度下(如0.1 nmol.L-1)即可强烈的诱导细胞发生凋亡,且凋亡的比率随着蛋白浓度的升高而增加。结论本实验制备的强化融合蛋白LDP-Hr-AE可以特异的与人表皮生长因子受体2结合,对肿瘤细胞具有强烈的杀伤活性和诱OBJECTIVE To construct a novel fusion protein consisting of oligopeptides specific for human epidermal growth fac- tor receptor 2 （HER2） and lidamycin （LDM） , and investigate its antitumor activity. METHODS Coding sequences of oligopeptides from complementarity determining region 3 （ CDR3 ） of anti-HER2 antibody C6. 5 heavy chain was fused to apoprotein of lidamycin to obtain the fusion gene ldp-Hr. Fusion protein LDP-Hr was expressed in E. coli and purified by affinity chromatography. The purity of LDP-Hr was analyzed by high HPLC. Immunofluorescenee assay and flow cytometry-based binding assay were used to investigate the binding activity of LDP-Hr to HER2 overexpressed cancer cells. The energized fusion protein LDP-Hr-AE was prepared by integrating the active enediyne chromophore （AE） of lidamycin into the LDP-Hr protein. MTI＂ assay was used to measure the in vitro cytotoxicity of LDP-Hr-AE and Annexin V-FITC/PI staining assay was used to analyze its apoptosis-inducing efficacy. RESULTS Fusion protein LDP-Hr was constructed con＇ecfly and expressed in E. coli in a secretory manner. The production of LDP-Hr was 40 mg per liter fer- mentation broth, and the purity of fusion protein was 97. 4% as analyzed by HPLC. LDP-Hr showed strong binding activity to cancercells highly expressing HER2, such as SK-BR-3 and SK-OV-3 cells. The energized fusion protein LDP-Hr-AE exhibited more potent cytotoxicity to SK-BR-3 and SK-OV-3 cells than LDM as measured by MTT assay. The results from Annexin V-FITC/PI staining assay also revealed that LDP-Hr-AE significantly induced cell apoptosis even at very low concentrations. CONCLUSION The novel ener- gized fusion protein LDP-Hr-AE bounds to HER2 specifically, and shows potent cytotoxicity and apoptosis-inducing activity to cancer cells, which suggests that it would be a promising candidate for targeted cancer therapy.

关 键 词：人表皮生长因子受体2 融合蛋白 力达霉素 细胞凋亡 抗肿瘤 

分 类 号：R737.9[医药卫生—肿瘤]