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抗Her-2抗体与人β防御素Ⅱ融合蛋白的表达、纯化及活性鉴定

Expression,purification and activity assay of SUMO-HBD2-4D5 fusion protein
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摘要 利用小分子泛素样修饰蛋白(small ubiquitin-like modifier,SUMO)融合表达系统,在大肠杆菌Origami B(DE3)中表达人β防御素Ⅱ(humanβdefensin 2,HBD2)与抗人表皮生长因子受体2(Her-2)单链抗体(4D5 scFv)的融合蛋白。经20℃,l mmol/L IPTG诱导24 h后,获得相对分子质量约为41 kD的SUMO-HBD2-4D5融合蛋白,为可溶性表达,表达量占菌体上清总蛋白的42.2%。利用组氨酸亲和凝胶色谱(Ni-NTAsepharose)、特异性SUMO蛋白酶(SUMO protease)酶切和分子筛(Sephadex G-25)联用的方法可获得纯度大于95%的目的蛋白HBD2-4D5,其最终得率达到15 mg/L。HBD2-4D5 200μg对金黄色葡萄球菌(ATCC25923)和大肠杆菌K12D31具有较明显的杀伤作用。免疫荧光分析结果显示,HBD2-4D5蛋白能结合于Her-2高表达的人乳腺癌细胞SK-BR-3表面。本实验表达的HBD2-4D5双功能蛋白具有较好的抗菌活性和特异性结合SK-BR-3表面Her-2的能力,为日后用于靶向治疗Her-2高表达的肿瘤疾病提供研究基础。 Human beta defensin 2（HBD2） is a small antimicrobial peptide,which is able to inhibit the proliferation of multiple cancer cells.In this study,HBD2 was linked to a single chain of the Her-2 antibody fragment（4D5 scFv） by genetic engineering,and the soluble HBD2-4D5 was expressed successfully by the small ubiquitin-like modifier（SUMO）molecular chaperones in E.coli.HBD-4D5 was purified using the combination of Ni-NTA Sepharose FF,digestion by specific SUMO protease and Sephadex G-25,the purity was higher than 95% with a yield of 15 mg/L.The fusion HBD2-4D5 showed significant antibacterial activity against E.coli K12D31 and Staphylococcus aureus（ATCC25923）,suggesting that the activity of HBD2 was reserved.Also,HBD2-4D5 demonstrated selective binding activity to SK-BR-3,a Her-2 over expressed breast cancer cell line.This finding provides foundations for further investigation for the treatment of the HER-2 over expressed breast cancer.
作者 袁丰瑞 张敏静 仝雷 苏志坚 项琪 黄亚东
机构地区 暨南大学医药生物技术研究开发中心 暨南大学广东省生物工程药物重点实验室 暨南大学基因工程药物国家工程研究中心
出处 《中国药科大学学报》 CAS CSCD 北大核心 2011年第3期266-271,共6页 Journal of China Pharmaceutical University
基金 广东省中国科学院全面战略合作项目(No.2010B090301016) 广东省教育厅产学研重大项目(No.cgzhzd0710) 温州市科技支撑计划资助项目(No.Y20090279)~~
关键词 人β防御素Ⅱ 人表皮生长因子受体2 融合蛋白 靶向作用 HBD2 Her-2 fusion protein target effects
分类号 Q784 [生物学—分子生物学] R966 [医药卫生—药理学]
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参考文献16

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中国药科大学学报
2011年 第3期

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