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  

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

作　　者：沈涛[1] 李妍[2] 杨蕾[1] 郭洲洋 巴根[1] 郭然[1] 陈之光[1] 付勤[1] 

机构地区：[1]中国医科大学附属盛京医院骨科,辽宁沈阳110004 [2]中国医科大学基础医学院细胞生物学教研室,细胞生物学卫生部重点实验室,辽宁沈阳110001

出　　处：《现代肿瘤医学》2017年第12期1859-1862,共4页Journal of Modern Oncology

基　　金：国家自然科学基金青年基金(编号:31201053);国家自然科学基金面上项目(编号:81372337)

摘　　要：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c DNA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c DNA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。Objective： To construct the expression plasmid of human sorbin and SH3 domain containing 1 （ hPlk4 ） gene and identify expression and localization of hPlk4 in U20S osteosarcoma cells. Methods ：Using pGEX - 4 T - 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 U20S osteosarcoma cells. The 3 ＊ Flag - hPlk4 plasmid expression in U20S cells was detected by Western blot assay. The localization of 3 ＊ Flag - hPlk4 fusion protein in U20S cells was observed with laser scanning confocal microscopy. The hPlk4 protein was purified by immunoprecipitation assay. Re-sults ：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 U20S 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.

关 键 词：hPlk4 WESTERN BLOT 免疫沉淀 骨肉瘤 

分 类 号：R738.1[医药卫生—肿瘤]