嵌合分子CblN/Grb2构建及其原核表达

Construction of chimeric CblN/Grb2 and its expression in prokaryon

目的构建CblN/Grb2嵌合分子,表达和纯化GST-CblN/Grb2融合蛋白,并考察嵌合分子CblN/Grb2在体外是否具有泛素连接酶活性。方法提取SKBR-3细胞的总RNA并反转录为cDNA,作为模板用PCR扩增Grb2基因的SH2片段;含有人全长CblcDNA的质粒pEFHACbl作为模板扩增Cbl基因N-端(CblN);用重叠延伸PCR方法,在CblN内部SH2两端引入BamHⅠ和EcoRV酶切位点并克隆入pcDNA3·1(+)。再以Grb2基因的SH2置换CblN的SH2即成为pcDNA3·1(+)-CblN/Grb2;以其为模板,通过PCR方法扩增CblN/Grb2并将其亚克隆至表达载体pGEX-4T-2,转化大肠杆菌,IPTG诱导表达GST-CblN/Grb2融合蛋白并用GlutathioneSepharose4B进行纯化;通过体外泛素化实验检测GST-CblN/Grb2是否具有泛素连接酶活性。结果成功构建了pGEX-4T-2-CblN/Grb2融合表达载体;在大肠杆菌DH5α中表达了GST-CblN/Grb2融合蛋白并获得了纯化蛋白;体外泛素化实验表明GST-CblN/Grb2具有泛素连接酶活性。结论GST-CblN/Grb2的表达、纯化及其体外活性的鉴定为进一步研究该嵌合泛素连接酶对HER2阳性肿瘤细胞生长的影响奠定了基础。

Objective To construct the pGEX-4T-2-CblN/Grb2, express and purify the corresponding chimeric protein; To investigate whether the chimeric CblN/Grb2 possesses ubiquitin ligase activity. Methods Total RNA of SK-BR-3 cells were isolated and reversely transcribed into cDNA, which were used as templates to amplify Grb2 SH2 by PCR. The gene fragment encoding the N-terminal part of Cbl protein （named as CblN） was amplified by PCR using pEFHACbl plasrnid encoding human Cbl as templates. BamH Ⅰ and EcoR Ⅴ restriction enzyme digestion sites were introduced into both flanks of SH2 by overlapping extension PCR and the modified gene were cloned into pcDNA3. 1（＋）. The pcDNA3.1（＋）-CblN/Grb2 was obtained by replacing SH2 of CblN with Grb2SH2 and then used as templates to amplify CblN/Grb2 by PCR. The pGEX-4T-2-CblN/Grb2 was constructed by subcloning CblN/Grb2 into the prokaryotic expressing vector pGEX-4T-2. The GST-CblN/Grb2 fusion protein was expressed in E. coli of DH5α under IPTG induction and further purified with Glutathione Sepharose 4B. In vitro ubiquitination assay was performed to investigate whether the GST-CblN/Grb2 fusion protein is able to mediate auto-ubiquitinating reaction, namely whether it possesses ubiquitin ligase activity. Results The fusion expressing vector of pGEX- 4T-2-CblN/Grb2 was successfully constructed; The GST-CblN/Grb2 fusion protein was correctly expressed and purified; In vitro ubiquitination assay indicated that GST-CblN/Grb2 fusion protein is able to mediate auto-ubiquitinating reaction and therefore possesses ubiquitin ligase activity. Conclusion Expression, purification of GST-CblN/Grb2 and identification of its‘ activity have laid the foundation for further study of chimeric ubiquitin ligase＇ effects on the growth of HER2 positive tumor cells.