重组泛素连接酶SH2-U—box／RING的构建与表达  

Construction and Expression of Recombinant Ubiquitin ligase SH2-U- box/RING

作　　者：茹懿[1] 李瑗春[1] 王秦豪[1] 钟代星[1] 李霞[1] 

机构地区：[1]第四军医大学基础部生物化学与分子生物学教研室肿瘤生物学国家重点实验室,西安市710032

出　　处：《医学分子生物学杂志》2012年第1期6-10,共5页Journal of Medical Molecular Biology

基　　金：国家自然科学基金（No.30800492,30873003,30972723）

摘　　要：目的构建重组泛素连接酶SH2-U—box、SH2-RING，并克隆进入pFlag—CMV4真核表达载体，为研究靶向降解慢性粒细胞白血病（chronic myelocytic leukemia，CML）患者瘤细胞中过度活化的BCR／ABL，抑制肿瘤细胞的生长提供基础。方法设计引物，扩增接头分子Grb2的SH2结构域以及E3泛素连接酶CHIP的U—box、Cb1的RING结构域，通过重组PCR，将SH2分别与U—box、RING进行融合，融合片段双酶切之后插入真核表达载体pFlag—CMV4，经过酶切鉴定及测序后，转染HEK293T细胞，Western印迹验证重组质粒的表达。结果PCR结果提示SH2-U—box条带大小888bp，SH2一RING大小为633bp，重组质粒酶切鉴定和测序结果均正确，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-SH2-U—box和pFlag—CMV4-SH2-RING，转染HEK293T细胞后能够正确表达，为后续研究奠定了基础。Objective To construct recombinant ubiquitin ligases SH2-U-box and SH2-RING into a eukaryotic expression vector pFLAG-CMV4, for further study of targeted degradation of BCR/ ABL excessively expressed in CML cells of and subsequent inhibition of the tumor cells. Methods SH2 domain of Grb2, U-box domain of CHIP and RING domain of Cbl were amplified with designed primers by routine PCR respectively. The SH2 domain fragment was then fused with U-box or RING domain fragment by recombinant PCR. The recombinant fragments were digested using double restric- tion enzymes EcoR I/EcoR V and inserted into the eukaryotic expression vector pFLAG-CMV4, and identified by enzyme digestion and sequencing. The constructed recombination plasmid was transfect- ed into HEK293T cells to verify the expression by Western-blot. Results PCR results showed the SH2-U-box and SH2-RING bands at size of 888bp and 633bp respectively. The constructed recombi- nation plasmids were verified by enzyme digestion and DNA sequencing. The fusion proteins were ex- pressed in HEK293T cell after transfection. Conclusion The eukaryotic expression vectors pFLAG- CMV4-SH2-U-box and pFLAG-CMV4-SH2-RING were successfully constructed, and confirmed to express correctly in HEK293T cell.

关 键 词：泛素 慢性粒细胞白血病 重组质粒 

分 类 号：Q783[生物学—分子生物学]