人IL-6/sIL-6R结合分子模型的建立及在药物筛选中的应用

Establishment of hIL-6 Protein Binding to sIL-6R Model for Screening IL-6 Inhibitors

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-6antibody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-6R1μg/well,IL-6500ng/well,IL-6antibody1μg/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。

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 BI21 （DE3） and the expressed protein was detected by Western blot assay and ELISA; Human slL-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- 6R1 （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.