SIP1在人舌癌Tca8113细胞中的定位和融合蛋白表达鉴定的研究

摘要

目的 检测Smad相互作用蛋白1(SIP1)在人舌癌Tca8113细胞系中的定位及融合蛋白的表达鉴定.方法 PCR扩增SIP1编码序列的全长,克隆到pCMV-Flag-MAT-Tag 1载体.利用共聚焦激光扫描显微镜观察SIP1和SIP1-Flag在人舌癌Tca8113细胞系中定位,并将构建好的Flag标签SIP1(SIP1 -Flag)真核表达载体转染到人舌癌Tca8113细胞系中,提取蛋白进行Western blot检测,利用Flag标签抗体进行免疫沉淀纯化SIP1-Flag融合蛋白.结果 构建了SIP1-Flag真核表达载体.SIP1和SIP1-Flag融合蛋白主要定位于人舌癌Tca8113的细胞核中,Western blot检测到SIP1-Flag融合蛋白表达,且在人舌癌Tca8113细胞中成功纯化SIP1-Flag蛋白.结论 成功构建真核表达载体.SIP1和SIP1-Flag主要定位于细胞核,成功鉴定融合蛋白表达并纯化SIP1-Flag融合蛋白.%Objective To detect the localization of Smad interacting protein 1 ( SIP1) in human TcaSl 13 tongue cancer cells and the i-dentification of its recombinant protein expression. Methods The SIP1 coding sequence was totally amplified by polymerase chain reaction (PCR) method,and was subcloned into pCMV-Flag-MAT-Tag 1 vector. The localization of SIP1 and SIPl-Flag in Tca8113 cells were observed by laser scanning confocal microscopy. Recombinant plasmid containing SIPl-Flag was transfected into Tca8113 cells. Expression of the SIPl-Flag in Tca8113 cells was detected by Western blot. The SIPl-Flag fusion proteins were purified by immunoprecipitation assay. Results Construction of eukaryotic expression plasmid of SIP1 -Flag gene was done. SIP1 and SIP1 -Flag were mainly localized in the nucleus of Tca8113 cells. Expression and purification of SIPl-Flag fusion proteins in Tca8113 cells were detected by Western blot. Conclusions The recombinant plasmids were successfully cloned into eukaryotic expressing vector. The SIP1 protein and SIPl-Flag were localized in the nucleus of Tca8113 cells. Expression and purification of recombinant SIPl-Flag proteins were identified.