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目的构建在哺乳动物细胞中表达的p38丝裂原活化蛋白激酶(MAPK)红色荧光蛋白(RFP)融合表达载体。方法将克隆在pcDNA3上的FLAG标记的p38MAPK亚克隆到红色荧光蛋白载体pDsRed1-N1上,随后转染HeLa细胞。在荧光显微镜下观察结果。结果重组质粒经酶切、PCR和测序证明正确无误,并在HeLa细胞中得到高量表达。融合蛋白发出的红色荧光表明p38MAPK弥散分布于细胞质中,细胞核中也有一定的分布。结论成功构建了p38MAPK红色荧光蛋白融合载体,该载体能在哺乳动物细胞中进行表达,为研究p38MAPK的细胞内定位提供了一个重要的工具。
Objective To construct a p38 mitogen - activated protein kinase (MAPK) red fluorescent protein (RFP) fusion expression vector expressed in mammalian cells. Methods The FLAG-labeled p38MAPK cloned on pcDNA3 was subcloned into the red fluorescent protein vector pDsRed1-N1 and then transfected into HeLa cells. Observe the results under a fluorescence microscope. Results The recombinant plasmids were verified by restriction enzyme digestion, PCR and sequencing. The recombinant plasmids were highly expressed in HeLa cells. The red fluorescence of the fusion protein indicates that p38MAPK is diffusely distributed in the cytoplasm and has a certain distribution in the nucleus. Conclusion The p38MAPK red fluorescent protein fusion vector was successfully constructed, which can be expressed in mammalian cells. It provides an important tool for studying the intracellular localization of p38MAPK.