中文摘要：

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。

英文摘要：

Objective To detect the localization of Smad interacting protein 1（SIP1） in human Tca8113 tongue cancer cells and the identification 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 SIP1-Flag in Tca8113 cells were observed by laser scanning confocal microscopy.Recombinant plasmid containing SIP1-Flag was transfected into Tca8113 cells.Expression of the SIP1-Flag in Tca8113 cells was detected by Western blot.The SIP1-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 SIP1-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 SIP1-Flag were localized in the nucleus of Tca8113 cells.Expression and purification of recombinant SIP1-Flag proteins were identified.