重组人IL-17A和IL-17F的制备方法及活性测定

Preparation of Recombinant Human IL-17A and IL-17F and Determination of Their Bioactivities

人IL-17A和IL-17F具有很高的同源性,在炎症性疾病、自身免疫性疾病和肿瘤中都发挥着重要的作用,是当前研究的热点。应用原核表达系统在大肠杆菌BL21(DE3)中高效表达了人IL-17A和IL-17F;经培养条件的优化,未发现可溶性目的蛋白的表达,免疫印记分析显示,重组蛋白位于包涵体中;对包涵体进行洗涤、凝胶过滤层析纯化和柱上复性,获得重折叠的可溶性蛋白;随后用SDS-PAGE对蛋白样品进行了纯度分析、采用免疫印记和质谱的方法鉴定蛋白产物成分、用MC3T3-E1和RAW264.7两个细胞株对IL-17A、IL-17F的生物学活性进行测定。结果显示,柱上复性的方法制备的该重组蛋白具有较高的纯度和活性。建立的重组人IL-17A和IL-17F的制备方法可为相关研究中细胞因子的大量应用提供参考。

Human IL-17A and IL-17F,two most important cytokines in inflame diseases,autoimmune diseases and cancer,have been studied extensively.For further research of their roles in Rheumatoid Arthritis（RA）,IL-17A and IL-17F were expressed in E.coli and purification protocol was established.Both the IL-17A and IL-17F were expressed in E.coli BL21（DE3）in high yield in the form of inclusion bodies.The inclusion bodies were washed in PBS,TritonX-100,4 mol/L Urea subsequently,and then dissolved in lysis buffer.The denatured target proteins in the supernatant were purified by gel filtration chromatography.The purified denatured target proteins were subjected to on-column refolding.The refolded proteins,with purity of more than 95% on SDS-PAGE,were target protein confirmed by MS and Western blotting.The biological effects of refolded IL-17A and IL-17F were evaluated by enzyme-linked immunosorbent assay（ELISA）in different cell lines.The expressions of NO in MC3T3-E1 and MMP-9 in RAW264.7 were increased,which indicated that the recombination proteins we purified have the biological activities.