3*Flag-hPlk4真核表达载体的构建及其在骨肉瘤细胞中的表达和定位

目的:构建3*Flag-hPlk4真核表达载体,并证实重组表达载体在骨肉瘤细胞内的表达及定位.方法:以pGEX-4T-2-hPlk4为模板,利用聚合酶链反应扩增hPlk4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.进一步将构建的重组载体进行双酶切和测序鉴定,并转染到U2OS骨肉瘤细胞中,Western blot检测重组蛋白的表达.同时利用共聚焦激光显微镜观察3*Flag-hPlk4表达载体在U2OS细胞中的定位,进一步利用免疫沉淀的方法纯化人源Plk4蛋白.结果:hPlk4基因cDNA全长成功构建到3*Flag真核表达载体中,Western blot检测到含有3*Flag的人源Plk4融合蛋白表达,进一步在U2OS骨肉瘤细胞中主要定位于细胞质和细胞核周,并成功纯化hPlk4蛋白.结论:成功构建了3*Flag-hPlk4真核表达质粒,同时鉴定了融合蛋白的表达,并成功纯化人源Plk4蛋白.3*Flag-hPlk4蛋白主要定位在细胞质和细胞核周.

Construction,expression and localization of 3*Flag-hPlk4 plasmid in osteosarcoma cells

Objective:To construct the expression plasmid of human sorbin and SH3 domain containing 1(hPlk4) gene and identify expression and localization of hPlk4 in U2OS osteosarcoma cells.Methods:Using pGEX-4T-2-hPlk4 plasmid as a template,human Plk4 coding sequence was amplified by polymerase chain reaction(PCR) and cloned into 3*Flag empty vector.After the target region was identified by double enzymes digestion and sequencing,the plasmid was transiently transfected into U2OS osteosarcoma cells.The 3*Flag-hPlk4 plasmid expression in U2OS cells was detected by Western blot assay.The localization of 3*Flag-hPlk4 fusion protein in U2OS cells was observed with laser scanning confocal microscopy.The hPlk4 protein was purified by immunoprecipitation assay.Results:Human Plk4 gene was successfully constructed into the 3*Flag expression vector.The expression of 3*Flag-hPlk4 fusion protein was detected by Western blot assay and pulled down by anti-flag antibody.Its localization is at the cytoplasm and perinuclear of U2OS cells.Conclusion:The 3*Flag-hPlk4 recombinant plasmid was successfully cloned into eukaryotic expression vector.The expression of 3*Flag-hPlk4 fusion protein was pulled down by anti-flag antibody and localized at the cytoplasm and perinucleus.