hPlk2基因真核表达载体的构建及蛋白的表达和定位-东南大学学报医学版

hPlk2基因真核表达载体的构建及蛋白的表达和定位

单位：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;
3. 明尼苏达大学实验病理系, 美国明尼苏达州明尼艾波利斯市 55455

关键词：人Polo样激酶2 Western blot 绿色荧光蛋白质粒构建

分类号：Q291; Q786; Q591.2

出版年·卷·期（页码）：2012·31·第二期（162-165）

摘要：

目的:构建人Polo样激酶2(hPlk2)真核表达载体并证实该融合蛋白在细胞内的表达及定位。方法:提取工具细胞Hela的mRNA,反转录为cDNA。PCR扩增hPlk2基因cDNA全长,并将其亚克隆至pEGFP-C1表达载体中。然后将构建的重组质粒进行酶切和测序鉴定,并转染到工具细胞COS-7中,提取细胞蛋白进行Western blot检测。最后利用激光扫描共聚焦显微镜观察pEGFP-hPlk2在COS-7细胞内的定位。结果:hPlk2基因cDNA全长克隆到了真核表达载体pEGFP-C1中,酶切鉴定片段大小为2058bp,并测序成功。Western blot检测到了pEGFP-hPlk2融合蛋白表达,分子量约为105kDa。pEGFP-hPlk2在COS-7细胞中主要定位于细胞质和核周。结论:成功构建了hPlk2基因cDNA全长的真核表达载体,pEGFP-hPlk2蛋白在COS-7细胞中主要定位于细胞质和核周。

Objective: To construct the expression plasmid of human Polo-like kinase 2 (hPlk2) gene and identify the expression and localization of the fusion protein. Methods: Total mRNA was extracted from Hela cells, and cDNA was synthesized by reverse transcription. The hPlk2 coding sequence was amplified by polymerase chain reaction (PCR) and subcloned into pEGFP-C1 vector. After the target region was identified by restriction enzyme digestion and sequencing, the plasmid was transfected into COS-7 cells. The expression of the recombinant plasmid in COS-7 cells was detected by Western blot assay. The localization of pEGFP-hPlk2 in COS-7 cells was observed with laser scanning confocal microscopy. Results: hPlk2 was constructed into the expressing vector pEGFP-C1 successfully. The length of the fragment identified by restriction enzyme digestion was 2058bp. The expression of pEGFP-hPlk2 fusion protein with a molecular weight of 105kDa was detected by Western blot. The pEGFP-hPlk2 fusion protein was mostly localized in the cytoplasm and perinucleus of COS-7 cells. Conclusion: The recombinant plasmid of hPlk2 gene was successfully cloned into eukaryotic expressing vector, and the pEGFP-hPlk2 fusion protein was mostly localized in the cytoplasm and perinucleus of COS-7 cells.

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