骨肉瘤MG-63细胞中Polo样激酶2的表达和定位

Expression and localization of Polo-like kinase 2 in osteosarcoma cells

目的:构建真核表达载体*3 Flag-hPlk2并检测其在骨肉瘤细胞内的表达及定位。方法:以GST-hPlk2为模板,利用PCR扩增hPlk2基因cDNA全长,并将其克隆至含有*3 Flag标签的真核表达载体中。将构建的重组质粒进行酶切和测序鉴定,并转染到骨肉瘤细胞MG-63中,提取细胞蛋白进行Western blot检测。利用共聚焦激光扫描显微镜观察*3 Flag-hPlk2在MG-63细胞中的定位,免疫沉淀法纯化hPlk2蛋白。结果:hPlk2基因cDNA全长成功构建到真核表达载体*3 Flag中,Western blot检测到*3 Flag-hPlk2融合蛋白表达,分子量约为80kDa。*3 Flag-hPlk2在骨肉瘤MG-63细胞中主要定位于细胞质和核周,并成功纯化hPlk2蛋白。结论:成功构建了*3 Flag-hPlk2真核表达质粒,同时鉴定了*3 Flag-hPlk2融合蛋白的表达,并纯化hPlk2蛋白。*3 Flag-hPlk2蛋白主要定位在细胞质和核周。

Objective：To construct the expression plasmid of human Polo-like kinase 2（hPlk2） gene and identify the expression and localization of hPlk2 in osteosarcoma MG-63 cells.Methods： Human Plk2 coding sequence was obtained by polymerase chain reaction（PCR） amplification and cloned into 3＊ Flag vector.After the target region was identified by enzyme digestion and sequencing,the plasmid was transfected into osteosarcoma MG-63 cells.The expression of the recombinant plasmid in MG-63 cells was detected by Western blot.The localization of 3＊ Flag-hPlk2 in MG-63 cells was observed with laser scanning confocal microscopy.Purify the hPlk2 protein by immunoprecipitation assay.Results： hPlk2 was constructed into the expressing vector 3＊ Flag successfully.The length of the fragment identified by restriction enzyme digestion was 2058bp.The expression of 3＊ Flag-hPlk2 fusion protein with a molecular weight of 80kDa was detected by Western blot and pulled down by Flag antibody,and its localization was in the cytoplasm and perinucleus in MG-63 cells.Conclusion： The recombinant plasmid of hPlk2 gene was successfully cloned into eukaryotic expressing vector,and the expression of 3＊ Flag-hPlk2 fusion protein was identified and pulled down by Flag antibody,expressed in cytoplasm and perinucleus.