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目的 构建真核表达载体GFP-hPIk1并检测在骨肉瘤细胞内的表达及定位.方法 以pcDNA3.1-hPlk1为模板,利用聚合酶链反应扩增hPlk1基因的cDNA全长,并将其克隆至pEGFP-C1真核表达载体中.进一步将构建的重组质粒进行酶切和测序鉴定,并转染到U2OS骨肉瘤细胞中,提取细胞总蛋白进行Western blot 检测.同时利用激光共聚焦扫描显微镜观察GFP-hPlk1在U2OS细胞中的定位,免疫沉淀的方法纯化hPlk1蛋白.结果 hPlk1基因cDNA全长成功构建到真核表达载体pEGFP-Cl中,Western blot检测到了GFP-hPlk1融合蛋白表达,分子质量Mr约为93 000Da.GFP-hPlk1在骨肉瘤细胞U2OS中主要定位于细胞质和核周,并成功纯化hPlk1蛋白.结论 成功地构建了GFP-hPlk1真核表达质粒,并在骨肉瘤细胞U2OS中表达.“,”Objective To construct an eukaryotic expression vector GFP-hPlk1 and identify the expression and localization of hPlkl in osteosarcoma U2OS cells.Methods Using pcDNA3.1-hPlk1 as a template,we obtained human Plk1 coding sequence by polymerase chain reaction(PCR) amplification and cloned it into the eukaryotic expression vector.The plasmid was identified by restriction enzyme digestion and DNA sequencing,GFP-hPlk1 was transiently transfected into osteosarcoma U2OS cells and examined by Western blot.The localization of GFP-hPlk1 in U2OS cells was observed with laser scanning confocal microscopy.hPlk1 protein was purified by immunoprecipitation assay.Results hPlk1 cDNA was successfully constructed into the expressing vector pEGFP-C 1.The length of the fragment identified by restriction enzyme digestion was 1812 bp.The expression of GFP-hPlk1 fusion protein with a molecular weight of 93 000Da was detected by Western blot and pulled down by GFP antibody,localized in the cytoplasm and perinucleus in U2OS cells.Conclusion The recombinant plasmid of hPlk1 gene was successfully constructed,GFP-hPlk 1 fusion protein was expressed in U2OS cells.