人白细胞介素2受体β(IL-2RB)基因真核表达及与JAK1的相互作用的检测  

Expression of eukaryotic vector human Interleukin 2 receptorβ(IL-2RB) and detection with interaction protein JAK1

作　　者：郭欣欣[1] 邓巧亭 王刚[1] 吴涵韬 李坤[1] 吴英松[1] 刘天才[1] 

机构地区：[1]南方医科大学检验与生物技术学院抗体工程研究所,广东广州510515

出　　处：《生物技术》2017年第1期29-33,共5页Biotechnology

基　　金：国家高技术研究发展计划(863计划)项目("蛋白质相互作用均相发光仪器和配套试剂研究";No.2014AA020904);国家自然科学基金项目("基于能量转移的新型免疫荧光传感器研究及其在高通量分析蛋白-蛋白相互作用中的初步应用";No.21575058;"一种新型量子点均相荧光免疫分析方法的建立及其初步应用";No.81271931)

摘　　要：[目的]构建含人白介素2受体β(IL-2RB)基因的真核表达质粒p ENTER-IL-2RB-His,并在293T中进行真核表达,并用免疫共沉淀检测JAK1与IL-2RB在细胞内的相互作用。[方法]在Hela细胞中提取人总RNA,通过RT-PCR获得人IL-2RB的基因全长,并将其克隆至真核表达载体p ENTER-His中。经PCR克隆,双酶切、测序鉴定后,将重组质粒p ENTER-IL-2RB-His转染至293T细胞中。免疫印迹法检测不同时间点的IL2RΒ蛋白(24 h、36 h、48 h)在293T细胞中的表达,用免疫共沉淀检测JAK1和IL-2RB蛋白之间的相互作用。[结果]经PCR克隆、双酶切、测序鉴定质粒克隆正确。免疫印迹可见61 k Da的目的蛋白。共同转染JAK1和IL-2RB的质粒,免疫印迹可见分别为133 k Da和61 k Da的目的条带。[结论]成功获得IL-2RB基因全长,成功构建p ENTER-IL-2RB-His真核表达质粒,并在293T细胞中成功表达,随着时间的推移其表达量增高。免疫共沉淀可以检测到JAK1和IL-2RB两者的蛋白相互作用,这为下一步研究JAK1和IL-2RB这一对蛋白的相互作用的作用方式及作用机制奠定了基础。[Objective] To construct a eukaryotic express vector p ENTER-IL-2RB-His,which can express human Interleukin 2 receptor beta（IL-2RB） in 293 T cells,then to detect the intracellular interaction between JAK1 and IL-2RB by co-immunoprecipitation. [Methods] Human total RNA was extracted from Hela cells,then IL-2RB gene was amplified by RT-PCR and inserted into p ENTER-His. 293 T cells were transfected with the recombined plasmid p ENTER-IL-2RB-His.The express level of IL-2RB at 24 hours,36 hours and 48 hours was determined by Western Blotting. The determination of interaction proteins between JAK1 and IL-2RB was verified by Co-immunoprecipitation[Results] Restriction analysis and sequencing proved the accuracy of the recombinant plasmid,and the target protein band was identified by Western Blotting. When293 T cells were transfected with JAK1 and IL-2RB recombined plasmid simultaneously,the target protein band in 133 k Da and 61 k Da were identified by Western Blotting. Co-immunoprecipitation could validate the interaction between JAK1 and IL-2RB. [Conclusion] Recombined plasmid p ENTER-IL-2RB-His was successfully constructed and expressed effectively in 293 T cells. The interaction between JAK1 and IL-2RB was validated by co-immunoprecipitation,which laid a foundation of mechanism research on protein-protein interaction of JAK1 and IL-2RB.

关 键 词：白介素2受体β(IL-2RB) 真核表达 免疫共沉淀 蛋白相互作用 

分 类 号：Q291[生物学—细胞生物学]