利用SIE转录活性荧光素酶报告基因系统检测白细胞介素6受体拮抗剂的生物活性

Detection of biological activity of interleukin-6 receptor antagonist by SIE transcriptionally active luciferase reporter gene system

目的:利用sis诱导元素(sis-inducible element,SIE)转录活性荧光素酶报告基因系统检测白细胞介素6(IL-6)受体拮抗剂的生物活性。方法:采用脂质体转染法将含有SIE转录因子组件及荧光素酶报告基因的质粒pGL 4.47转入293T细胞,并通过潮霉素压力筛选获得稳定转染细胞株。加入IL-6激活稳转细胞株中的荧光素酶报告基因,构建SIE转录活性荧光素酶报告基因系统,并进一步建立基于此系统的IL-6受体拮抗剂药物的生物学活性定量检测方法。结果:IL-6可激活293T-SIE稳定转染细胞中的荧光素酶报告基因,且具有量效关系。对此系统检测IL-6受体拮抗剂药物的生物学活性进行方法学验证,显示本检测方法准确性及重复性均较好。结论:建立了一种基于荧光素酶报告基因系统的IL-6受体拮抗剂药物生物学活性检测方法,与现有方法相比,具有耗时少、准确性高、重复性好的优势。

Objective：To determine bioactivity of interleukin-6（IL-6） receptor antagonists using sis-inducible element（SIE） transcriptionally active luciferase reporter gene system.Methods：Plasmid pGL 4.47 containing the SIE transcription factor and the luciferase reporter gene was transferred into 293T cells by liposome transfection, and the stable transfected cell line（293T-SIE） was obtained by hygromycin pressure screening.Being activated by IL-6, the luciferase reporter gene system was constructed and the bioactivity detection method of IL-6 receptor antagonist based on this system was further established.Results：In 293T-SIE, the luciferase reporter gene could be activated by IL-6 with a dose-effect relationship.The methodological validation of this system for the detection of biological activity of IL-6 receptor antagonists showed that the method had good accuracy and reproducibity.Conclusion：A novel method was established to detect the bioactivity of IL-6 receptor antagonist based on the luciferase reporter gene system which had the advantages of being less time-consuming, having higher accuracy and better reproducibility compared with the existing methods.