中文摘要：

目的构建人SORBSl真核表达载体并检测在骨肉瘤细胞内的表达及定位。方法以GST．hSORBSl为模板，利用PCR扩增hSORBSl基因cDNA全长，并将其克隆至3％Flag标签的真核表达载体中。进一步将构建的重组载体进行酶切和测序鉴定，并转染到骨肉瘤细胞MG-63中，提取蛋白进行Westernblot检测。同时利用共聚焦激光显微镜观察3SFlag-hSORBSl在MG-63细胞中的定位，使用免疫沉淀的方法纯化hSORBSl蛋白。结果hSORBSl基因cDNA全长成功构建到真核表达载体3SFlag中，Westernblot检测到3＊Flag．hSORBSl融合蛋白表达，且在骨肉瘤细胞MG一63中主要定位于细胞周边，并成功纯化hSORBSl蛋白。结论成功构建3SFlag．hSORBSl真核表达质粒，同时鉴定其融合蛋白的表达，并纯化hSORBSl蛋白。3％Rag-hSORBSl蛋白主要定位在细胞周边。

英文摘要：

Abstract： Objective To construct a eukaryotic vector of human sorbin and SH3 domain containing the 1 （ hSORBS1 ） gene and identify expression and localization of hSORBS1 in osteosarcoma MG-63 cells. Methods Using GSThSORBS1 as a template, we obtained the human SORBS1 coding sequence by polymerase chain reaction （PCR） ampli- fication and cloned it into the eukaryotic vector 3 ＊ Flag. The insert was identified by restriction enzyme digestion and DNA sequencing. 3 ＊ Flag-hSORBS1 was transfected into osteosarcoma MG-63 cells. Expression of the recombinant plasmid in MG-63 cells was detected by Western blot. The localization of 3 Flag-hSORBS1 in MG-63 cells was observed by laser scanning confocal microscopy. The hSORBS1 fusion proteins were purified by immunoprecipitation as- say. Results hSORBS1 was successfully constructed into the eukaryotic vector 3 ＊ Flag. Expression of 3 ＊ FIaghSORBS1 fusion protein was detected by Western blot, and it was localized at the periphery of MG-63 cells. Conclusion The eukaryotic expression plasmid of hSORBS1 was successfully constructed. Expression of fusion pro- teins was identified. The 3 ＊ Flag-hSORBS1 fusion protein was localized at the cell periphery.