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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2009, Vol. 29 Issue (11): 60-65    
技术与方法     
人IL-6 /sIL-6R 结合分子模型的建立及在药物筛选中的应用
史继静1,刘朝奇1*,邹坤2,杨祖伟2,高明星2,杨凡1
1.三峡大学分子生物学研究所 宜昌 443002
2.三峡大学天然产物研究与利用湖北省重点实验室 宜昌 443002
Establishment of hIL-6 Protein Binding to sIL-6R Model for Screening IL-6 Inhibitors
SHI Ji-jing1,LIU Zhao-qi1,ZOU Kun2,YANG Zu-wei2,GAO Ming-xing2,YANG Fan1
1.Institute of Molecular Biology, Three Gorges University, Yichang 443002, China
2.Hubei Provincial Key Laboratory of Natural Product Research and Development， Three Gorges University, Yichang 443002, China
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摘要：

目的：建立人IL-6 /sIL-6R 结合的分子模型，用于筛选IL-6 /sIL-6R的抑制剂。方法：将人IL-6基因克隆至原核表达载体pET28a（+）中表达IL-6蛋白，western blot及人IL-6检测试剂盒分析鉴定表达蛋白。同法将人sIL-6R在pET15b载体中表达，纯化并用western blot检测目的蛋白。依据ELISA原理建立IL-6 /sIL-6R 结合的分子模型，并通过改变IL-6、sIL-6R及IL-6 antibody的浓度来优化该模型，用于IL-6 /sIL-6R拮抗药物的筛选。结果：人IL-6可在载体PET28a（+）中高效表达，且经western blot鉴定正确，人IL-6检测试剂盒检测显示具有较高的免疫活性。sIL-6R在PET15b中表达，western blot鉴定正确。通过对IL-6 /sIL-6R结合的分子模型的优化，得到其最佳条件为：IL-6R 1?g/well, IL-6 500ng/well, IL-6 antibody 1?g/well。应用该模型筛选发现有些化合物可显著抑制IL-6与其受体的结合。结论：成功构建IL-6 /sIL-6R结合的分子模型，为高通量筛选IL-6拮抗剂提供平台。
关键词： IL-6;  sIL-6R;  蛋白表达;  ELISA;  药物筛选    
Abstract:

 AIM: To eatablish the model of hIL-6 protein binding to sIL-6R for screening IL-6 inhibitors. METHODS: Human IL-6 gene was cloned into pET28a(+) prokaryotic expressing vector. The recombinant protein IL-6 was induced by IPTG in E. coli BL21(DE3) and the expressed protein was detected by Western blot assay and ELISA; Human sIL-6R gene was cloned into pET15b prokaryotic expressing vector. The recombinant protein sIL-6R was induced by IPTG in E. coli BL21(DE3) and the expressed protein was detected by Western blot assay.Then IL-6/sIL-6R binding assay was established using enzyme-linked immunosorbent assay (ELISA), and optimized the model through changing the concentrations of IL-6, sIL-6R and IL-6 antibody. RESULTS: Human IL-6 and sIL-6R could be expressed in E. coli BL21(DE3) with high efficiency. The recombinant proteins were characterized by western blot and ELISA.Through optimizing the model,the best conditions were obtained: IL-6R 1?g/well, IL-6 500ng/well, IL-6 antibody 1?g/well.And some components detected by the model had significantly inhibitory effect on IL-6/sIL-6R. CONCLUSION: The screening model of hIL-6 protein binding to sIL-6R was successfully established,which provided a reliable platform for high throughput screening of IL-6 inhibitors.
Key words: IL-6    sIL-6R    protein expression    ELISA    drug screening
收稿日期: 2009-04-07 出版日期: 2009-12-07
ZTFLH:  Q789  
通讯作者: 刘朝奇     E-mail: chaoqil@yahoo.com
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史继静
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高明星
杨凡

引用本文:

史继静 刘朝奇 邹坤 杨祖伟 高明星 杨凡. 人IL-6 /sIL-6R 结合分子模型的建立及在药物筛选中的应用[J]. 中国生物工程杂志, 2009, 29(11): 60-65.

SHI Ji-Jing, LIU Chao-Ai, JU Kun, YANG Jie-Wei, GAO Meng-Xing, YANG Fan. Establishment of hIL-6 Protein Binding to sIL-6R Model for Screening IL-6 Inhibitors. China Biotechnology, 2009, 29(11): 60-65.

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https://manu60.magtech.com.cn/biotech/CN/    或    https://manu60.magtech.com.cn/biotech/CN/Y2009/V29/I11/60

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