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袁丰瑞, 张敏静, 仝雷, 苏志坚, 项琪, 黄亚东. 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[J]. 中国药科大学学报, 2011, 42(3): 266-271.
引用本文: 袁丰瑞, 张敏静, 仝雷, 苏志坚, 项琪, 黄亚东. 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[J]. 中国药科大学学报, 2011, 42(3): 266-271.
shu
YUAN Feng-rui, ZHANG Min-jing, TONG Lei, SU Zhi-jian, XIANG Qi, HUANG Ya-dong. Expression,purification and activity assay of SUMO-HBD2-4D5 fusion protein[J]. Journal of China Pharmaceutical University, 2011, 42(3): 266-271.
Citation: YUAN Feng-rui, ZHANG Min-jing, TONG Lei, SU Zhi-jian, XIANG Qi, HUANG Ya-dong. Expression,purification and activity assay of SUMO-HBD2-4D5 fusion protein[J]. Journal of China Pharmaceutical University, 2011, 42(3): 266-271.
shu

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

    摘要
  • 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（human β defensin 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-NTA sepharose）、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。

     

    Abstract: 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.coliK12D31 andStaphylococcus 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.

     

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