重组人白细胞介素-35的真核表达及生物活性  

Eukaryotic Expression and Biological Activity of Human Interleukin-35

作　　者：马娟[1] 谢良志[1,2] 

机构地区：[1]中国医学科学院北京协和医学院细胞工程中心,北京100005 [2]北京义翘神州生物技术有限公司,北京100176

出　　处：《中国医学科学院学报》2013年第6期618-622,共5页Acta Academiae Medicinae Sinicae

基　　金：十二五“重大新药创制”科技重大专项(2011ZX09506-003)~~

摘　　要：目的构建重组人白细胞介素-35[rhIL-35-IgG1(Fc)]蛋白的真核表达载体并表达纯化该蛋白,研究IL-35与其受体糖蛋白130(gp130)的结合作用。方法构建IL-35真核表达载体pSTEP2-IL35-LFc,转染到HEK293T细胞中,采用亲和色谱分离纯化获得重组IL-35蛋白,SDS-PAGE及Western blot法鉴定蛋白表达,ELISA法检测IL-35-Fc与其受体链gp130的结合作用,并在M1小鼠白血病细胞上研究IL-35对gp130的生物学作用。结果获得高纯度的人IL-35-Fc重组蛋白,ELISA法检测结果发现IL-35-Fc与其受体链gp130具有结合作用,并且IL-35在M1小鼠白血病细胞上对gp130具有中和作用。结论成功制备了具有生物活性的人IL-35-IgG1(Fc)重组蛋白,发现IL-35能够与其受体链gp130的结合,在M1小鼠白血病细胞上对gp130也具有中和作用。Objective To express and purify recombinant human interleukin-35 [IL-35-IgG1 （Fc）] in eukaryotic expression system and to study the interaction of IL-35 with gpl30 protein. Methods A mammalian expression vector, pSTEP2-IL35-LFe, was constructed and transfected into HEK293T cells. Then rhIL-35-IgG1 （Fc） was expressed and purified with protein A affinity chromatography, and was examined with SDS-PAGE and Western blot analysis. The binding of IL-35 to its receptor gpl30 was investigated using enzyme-linked immu- uosorbent assay （ELISA） . The biological effect of IL-35 on gpl30 was explored in M1 myeloid leukemia cells. Results rhIL-35-Fc with high purity on reduced SDS-PAGE was obtained. ELISA confirmed that IL-35-Fc was bound to gpl30 and neutralized the function of gpl30 in M1 myeloid leukemic cells. Conclusions High-pu- rity and biologically active rhIL-35-Fc protein successfully produced in this study. IL-35 binds to gpl30 and neu- tralizes its activity of in M1 myeloid leukemic ceils.

关 键 词：重组人白细胞介素-35 糖蛋白130 M1小鼠白血病细胞 

分 类 号：Q78[生物学—分子生物学]