重组泛素连接酶PTB-U-box/RING的构建与表达

目的 构建重组泛素连接酶PTB-U-box、PTB-RING,并克隆进入pFLAG-CMV4真核表达载体,为研究靶向降解受体型酪氨酸激酶IGF-IR,抑制肿瘤细胞生长提供基础.方法 分别设计引物,扩增接头分子IRS-1的PTB结构域以及E3泛素连接酶CHIP的U-box、Cbl的RING结构域,再利用重组PCR,将PTB分别与U-box、RING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HeLa细胞,Western 印迹验证重组质粒的表达.结果 PCR结果显示PTB-U-box条带大小840 bp,PTB-RING大小为620 bp,重组质粒经酶切鉴定和测序结果正确,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-PTB-U-box和 pFLAG-CMV4-PTB-RING,并且转染HeLa细胞后证实其能够正确表达.

Construction and Expression of Recombinant Ubiquitin Ligase PTB-U-box/RING

Objective To construct recombinant ubiquitin ligases PTB-U-box and PTB-RING in an eukaryotic expression vector pFLAG-CMV4, and to study targeted degradation of IGF-1R and the inhibitory effect of the degraded GF-1R on tumor cell growth. Methods The FFB domain of IRS-1, U-box domain of CHIP and RING domain of Cbl were amplified with designed primers respectively by routine PCR. The amplified PTB was fused with U-box or RING by recombinant PCR. The recombinant fragments were digested using double restriction enzymes and then inserted into the eukaryotic expression vectors pFLAG-CMV4. After enzyme digestion and sequencing analysis, the constructed recombination plasmids were transfected into HeLa cells. Expression of the recombinant plasmids was verified by Western-blot. Results PCR showed the sizes of 840 and 620bp respectively for PTB-U-box and PTB-RING bands, and sequencing verified the constructed recombination plasmids. The fusion proteins were expressed in HeLa cell after transfection. Conclusion The eukaryotic expression vectors pFLAG-CMV4-PTB-U-box and pFLAG-CMV4-PTB-RING were successfully constructed, and were expressed correctly in HeLa cell after cell transfection, which may be utilized for further studies.