野生型和突变型GDF5蛋白表达及亚细胞定位的研究

目的研究人生长分化因子5基因(GDF5)c.1118T>G(p.L373R)突变对GDF5蛋白在HEK-293细胞系表达及其亚细胞定位的影响。方法构建pDsRed1-N1-GDF5-WT和pDsRed1-N1-GDF5-L373R载体,分别转染HEK-293细胞,细胞培养72h后通过荧光显微镜观察比较野生型和突变型GDF5蛋白荧光分布情况。结果 GDF5蛋白在HEK-293细胞中成功表达,在荧光显微镜下观察到转染野生型和突变型GDF5的细胞胞浆均匀发出红色荧光,而空载体组的红色荧光则弥散于全细胞,荧光强度均匀一致。结论野生型和突变型GDF5蛋白均定位于HEK-293细胞质。

Expression and subcellular localization of wild-type and mutant GDF5 protein in HEK-293 cells

Objective To investigate the subcellular localization of human growth differentiation factor 5 gene(GDF5) c.1118T>G(p.L373R) mutant protein in HEK-293 cell line. Methods pDsRed1-N1-GDF5-WT and pDsRed1N1-GDF5-L373R were constructed , and the plasmids were transfected into HEK-293 cell line, 72 hours later,the subcellular localization of wild-type and mutant GDF5 proteins was observed under fluorescence microscope . Results The expression of GDF5 protein was detected in HEK-293 cells transfected with pDsRed1-N1-GDF5-WT and pDsRed1-N1-GDF5-L373R , the wild-type and mutant GDF5 proteins were 1ocalized in the cytoplasm with red fluorescence, while red fluorescence was detected in both the cytoplasm and nucleus of HEK-293 cells transfected with pDsRed1-N1. Conclusion Both the wildtype and mutant GDF5 proteins were localized in the cytoplasm of HEK-293 cells.