重组可溶性人IL-6的原核表达纯化和活性鉴定

发布时间：2018-03-31 20:10

本文选题：IL-　切入点：原核表达纯化　出处：《现代免疫学》2013年06期

【摘要】：使用大肠杆菌BL21(DE3)重组表达可溶性人IL-6并对其进行纯化和活性鉴定。以人激活T细胞总RNA为模板,逆转录PCR扩增人IL-6的基因片段并将其克隆到原核表达载体pET32a(+)中,重组质粒转化大肠杆菌BL21(DE3),优化表达条件获得可溶性表达目的蛋白。细菌裂解上清经镍金属螯和层析纯化,SDS-PAGE鉴定其分子量大小,western blot鉴定其免疫原性,并通过IL-6依赖的细胞株T1165分析重组蛋白的活性。成功构建了pET32a(+)/IL-6表达载体,并获得了可溶性表达的重组人IL-6蛋白。SDS-PAGE显示,其分子量约为21kD,可被抗IL-6抗体特异性识别,并且该重组蛋白可刺激IL-6依赖的细胞株T1165增殖,比活性与标准品一致,约为1×106 U/mg。本实验获得了可溶性高表达的重组人IL-6蛋白,为进一步研究人IL-6奠定了基础。
[Abstract]:The soluble human IL-6 was expressed and purified by E. coli BL21DE3. The total RNA of human activated T cells was used as template. The gene fragment of human IL-6 was amplified by reverse transcription PCR and cloned into the prokaryotic expression vector pET32a (). The recombinant plasmid was transformed into Escherichia coli BL21DE-3, and the soluble expression protein was obtained by optimizing the expression conditions. The bacterial lytic supernatant was purified by nickel metal chelate and chromatography. Its molecular weight was identified by western blot and its immunogenicity was identified. The activity of recombinant protein was analyzed by IL-6 dependent cell line T1165. The expression vector of pET32awas successfully constructed, and the soluble recombinant human IL-6 protein. SDS-PAGE showed that its molecular weight was about 21kDand could be specifically recognized by anti IL-6 antibody. Moreover, the recombinant protein could stimulate the proliferation of IL-6 dependent cell line T1165, and its specific activity was about 1 脳 10 6 Ur / mg. The recombinant human IL-6 protein with high soluble expression was obtained in this experiment, which laid a foundation for further study of human IL-6.
【作者单位】：上海交通大学药学院;聊城大学药学院;第二军医大学肿瘤研究所;
【分类号】：R392.1

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