过表达MDA-7/IL-24蛋白的亚细胞定位

目的了解标签蛋白GFP的位置对过表达的MDA-7/IL-24融合蛋白亚细胞定位的影响。方法分别构建在MDA-7/IL-24 cDNA的5'端和3'端带有GFP-tag的重组表达质粒pEGFP-C1-mda-7/IL-24和pEGFP-N3-mda-7/IL-24,瞬时转染猴胚肾Cos7细胞,48h后用Hoechst 33258染核,在荧光显微镜下观察GFP-MDA-7/IL-24融合蛋白在Cos7细胞的亚细胞分布。结果测序表明重组质粒pEGFP-C1-mda-7/IL-24和pEGFP-N3-mda-7/IL-24构建正确;分别转染Cos7细胞后,在氨基端带有GFP-tag的MDA-7/IL-24融合蛋白表达阳性细胞中,绿色荧光均匀分布于胞浆和胞核,但在羧基端带有GFP-tag的MDA-7/IL-24融合蛋白表达阳性细胞中,绿色荧光则分布于核膜外侧和胞浆。结论GFP-tag位置的不同对过表达融合蛋白MDA-7/IL-24的亚细胞定位有显著的影响。

Localization of overexpressed MDA-7/IL-24 protein

Objective To find out the role of GFP-tag on the localization of overexpressed MDA-7/IL-24 protein.Methods Recombinant plasmids pEGFP-C1-mda-7/IL-24 with GFP-tag at NH4-terminal and pEGFP-N3-mda-7/IL-24 with GFP-tag at COOH-terminal were constructed.After the recombinant plasmids were transfected transiently into monkey embryonic kidney cell of Cos7 for 48 hours,nucleus of Cos7 cells were dyed with Hoechst 33258.Distribution of the fusion proteins GFP-tag-MDA-7/IL-24 was observed under the fluorescence microscope.Results Sequential analysis proved that recombinant plasmids pEGFP-C1-mda-7/IL-24 and pEGFP-N3-mda-7/IL-24 were constructed successfully.In the positive cells for MDA-7/IL-24 fusion protein with GFP-tag at NH4-terminal,the GFP was found to be distributed equally both in the cytoplasm and the nucleus.The GFP in the positive cells for MDA-7/IL-24 fusion protein with GFP-tag at COOH-terminal was found to be localized around the outer part of the nuclear membrane and the cytoplasm.Conclusion The different position of GFP-tag in the fusion protein influenced the distribution of overexpressed MDA-7/IL-24 markedly.