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

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.