College of Veterinary Medicine, Yangzhou University, Yangzhou, Jiangsu, China; Ministry of Education Key Lab for Avian Preventive Medicine, Yangzhou University, Yangzhou, Jiangsu, China.
J Virol Methods. 2013 Dec;194(1-2):118-22. doi: 10.1016/j.jviromet.2013.08.014. Epub 2013 Aug 28.
Green fluorescent protein (GFP) used as a powerful marker of gene expression in vivo has so far been applied widely in studying the localizations and functions of protein in living cells. In this study, GFP-labeled assay was used to investigate the subcellular localization of matrix (M) protein of different virulence and genotype Newcastle disease virus (NDV) strains. The M protein of ten NDV strains fused with GFP (GFP-M) all showed nuclear-and-nucleolar localization throughout transfection, whereas that of the other two strains were observed in the nucleus and nucleolus early in transfection but in the cytoplasm late in transfection. In addition, mutations to the previously defined nuclear localization signal in the GFP-M fusion protein were studied as well. Single changes at positions 262 and 263 did not affect nuclear localization of M, while changing both of these arginine residues to asparagine caused re-localization of M mainly to the cytoplasm. The GFP-M was validated as a suitable system for studying the subcellular localization of M protein and could be used to assist us in further identifying the signal sequences responsible for the nucleolar localization and cytoplasmic localization of M protein.
绿色荧光蛋白（GFP）作为一种强大的基因表达标志物，已广泛应用于研究活细胞中蛋白质的定位和功能。本研究采用 GFP 标记法研究了不同毒力和基因型新城疫病毒（NDV）株基质（M）蛋白的亚细胞定位。10 株 NDV 株的 M 蛋白与 GFP 融合（GFP-M）后，在整个转染过程中均显示核仁和核定位，而另外两株则在转染早期在核仁和核内，在转染后期在细胞质内。此外，还研究了 GFP-M 融合蛋白中先前定义的核定位信号的突变。位置 262 和 263 的单个变化不影响 M 的核定位，而将这两个精氨酸残基均变为天冬酰胺则导致 M 主要重新定位到细胞质。GFP-M 被验证为研究 M 蛋白亚细胞定位的合适系统，并可用于协助我们进一步确定负责 M 蛋白核仁定位和细胞质定位的信号序列。
