骨肉瘤细胞中Polo样激酶3的表达和定位

目的:构建新型人源真核表达载体3* Flag-hPlk3并同时检测其在骨肉瘤细胞U2OS中的表达及定位.方法:以GST-hPlk3作为模板,利用聚合酶链反应扩增人源Plk3目的基因eDNA全长,并将其克隆到含有3* Flag标签的真核表达载体中.进一步将构建的重组质粒进行酶切和测序分析鉴定,并转染至U2OS骨肉瘤细胞系中,提取细胞总蛋白进行Western blot检测.同时利用荧光共聚焦激光扫描显微镜观察3*Flag-hPlk3在U2OS细胞系中的定位,进一步利用免疫沉淀方法纯化人源Plk3蛋白.结果:hPlk3基因cDNA全长成功构建于含有3* Flag标签的真核表达载体中,Western blot检测3*Flag-hPlk3融合蛋白表达,分子量约为74kDa.3 * Flag-hPlk3在骨肉瘤细胞系U2OS中主要定位于细胞质及核周,并成功纯化了hPlk3蛋白.结论:成功的构建了含有3* Flag标签的人源Plk3真核表达质粒,同时鉴定3*Flag-hPlk3融合蛋白的表达,最终成功纯化hPlk3蛋白.3*Flag-hPlk3蛋白主要定位在细胞质和核周.

Expression and localization of Polo-like kinase 3 in osteosarcoma cells

Objective:To construct the expression plasmid of human Polo-like kinase 3 (hPlk3) gene and to identify the expression and localization of hPlk3 in U2OS osteosarcoma cells.Methods:GST-hPlk3 was used plasmid as a template,human Plk3 coding sequence was obtained by polymerase chain reaction (PCR) amplification and cloned into 3 * Flag expression plasmid.After the target region was identified by enzyme digestion and sequencing,the plasmid was transiently transfected into osteosarcoma U2OS cells.The expression of the recombinant plasmid in U2OS cells was detected by Western blot.The localization of 3 * Flag-hPlk3 in U2OS cells was observed with laser scanning confocal microscopy.The hPlk3 protein was purified by immunoprecipitation assay.Results:Human Plk3 coding sequence was constructed into the expressing vector 3 * Flag successfully.The length of the PCR fragment identified by restriction enzyme digestion was 1 941bp.The expression of 3 * Flag-hPlk3 fusion protein was detected by Western blot with a molecular weight of 74kDa and pulled down by Flag antibody.The localization of recombinant protein is in the cytoplasm and perinucleus of U2OS cells.Conclusion:The recombinant plasmid of hPlk3 gene was successfully constructed into eukaryotic expressing plasmid.The expression of 3 * Flag-hPlk3 fusion protein was identified and pulled down by Flag antibody.Furthermore,it was localized in cytoplasm and perinucleus.