重组泛素连接酶tTRIP-U-box的构建与表达  

Construction and Expression of the Recombinant Ubiquitin Ligase tTRIP-U-box

作　　者：钟代星[1] 王秦豪[1] 茹懿[1] 药立波[1] 李霞[1] 

机构地区：[1]第四军医大学生物化学与分子生物学教研室,陕西西安710032

出　　处：《现代生物医学进展》2013年第27期5205-5208,共4页Progress in Modern Biomedicine

基　　金：国家自然科学基金项目(30800492;30972723)

摘　　要：目的:构建重组泛素连接酶tTRIP-U-box,并克隆进入pFLAG-CMV4真核表达载体,为研究通过靶向降解接头蛋白TRAF(TNF Receptor Associated Factor)家族蛋白,从而抑制破骨细胞活性提供基础。方法:分别设计引物,扩增tTRIP(TRAF-interacting protein)分子C端伸展的coiled-coiled结构域以及E 3泛素连接酶CHIP的U-box结构域,再利用重组PCR,将tTRIP与U-box进行融合,双酶切之后将其插入真核表达载体pFLAG-CMV4,经过酶切鉴定及测序后,转染HEK-293T细胞系,Western印迹验证重组质粒的表达。结果:PCR结果显示tTRIP截短分子和tTRIP-U-box条带大小分别为660bp和1188bp,重组质粒经酶切鉴定和测序证实正确,转染后可见融合蛋白的表达。结论:成功构建真核重组表达载体pFLAG-CMV4-tTRIP-U-box,并且转染HEK-293T细胞系后证实其能够正确表达。Objective： To construct recombinant ubiquitin ligase tTRIP-U-box, we cloned it into an eukaryotic vector pFLAG-CMV4 and investigated the targeted degradation of TRAFs and the inhibitory effect of TRAFs degradation on the function of osteoclast. Methods： The coiled-coiled domain of TRIP and the U-box domain of CHIP were amplified with designed primers respectively by routine PCR. The amplified coiled-coiled domain was fused with U-box domain by recombinant PCR. The recombinant fragment was digested using double restriction enzymes and then inserted into the eukaryotic expression vectors pFLAG-CMV4. After confirmation by enzyme diges- tion and sequencing analysis, the constructed recombination plasmids were transfected into HEK-293T cell lines. Expression of the re- combinant plasmids was verified by Western-blot. Results： PCR showed that the fragment of tTRIP and TRIP-U-box were 660 bp, 1188 bp in size; enzyme digestion and sequencing analysis further confirmed the recombination plasmids; the fusion proteins were expressed correctly in HEK-293T cell line after transfection. Conclusion： The eukaryotic expression vector pFLAG-CMV4-tTRIP-U-box was sue- cessfully constructed, and was able to express the fusion protein correctly in 293T cell lines after transfection.

关 键 词：重组泛素连接酶 破骨细胞 基因表达 

分 类 号：Q75[生物学—分子生物学] Q78