肿瘤转移抑制相关基因TMSG-1核仁定位信号序列的鉴定

Identification of nucleolar localization signal sequence of tumor metastasis suppressor gene-1

目的鉴定肿瘤转移抑制相关基因TMSG-1蛋白中潜在的特异性定位信号序列并探索其亚细胞定位机制。方法聚合酶链反应（PCR）扩增TMSG-1开放读码框全长及不同长度的截断片段，定向克隆于绿色荧光蛋白（GFP）表达质粒pEGFP—N1；各融合蛋白表达质粒转染人胚。肾细胞系HEK293细胞；转染48h后提取细胞总蛋白进行GFP的Western b1ot检测或用冷丙酮固定细胞后激光共聚焦显微镜观察融合蛋白的亚细胞定位。结果GFP分别融合TMSG-1全长蛋白（aa1-380）及其截断片段T1（aa1-70）、T2（aa1-128）、T3（aa129—380）、T4（aa71—128）、T5（aa71—179）和T6（aa71—380），Westernb1ot检测结果显示成功表达了各融合蛋白。激光共聚焦显微镜观察亚细胞定位显示融合蛋白T4（aa71—128）主要定位于细胞核内的核仁部位，融合蛋白T6（aa71—380）以细胞核内弥散分布为主，而TMSG—1全长融合蛋白及融合蛋白T1、T2、T3、T5则定位于胞质。进-步的序列缺失去除T4（aa71—128）羧基末端10个氨基酸得到截断片段T4△119-128，T4△119-128融合的GFP仍位于细胞核，但核仁内的绿色荧光信号明显减弱。结论肿瘤转移抑制相关基因TMSG-1存在潜在的核仁定位信号，位于aa119—128（RRRRNQDRPS），这一发现为深入研究TMSG-1的亚细胞定位及相关功能奠定了基础。

Objective To identify the putative specific localization signal sequence of tumor metastasis suppressor gene-1 （TMSG-1） and to explore the mechanism of subcellular localization of TMSG-1 protein. Methods Vectors expressing green fluorescence protein （GFP） tagged different TMSG-1 fragments were generated and transfected into human embryo kidney 293 （HEK293） cells. The expression of those fusion proteins was detected by Western blotting and their subcellular localizations were observed by laser eonfoeal microscope. Results GFP was fused with the native TMSG-1 （aa1-380 ） or different fragments including T1 （aal-70）, T2 （aal-128） ,T3 （aa129-380） , T4 （aa71-128）, T5 （aa71-179） and T6 （aa71- 380）. Anti-GFP Western blotting showed that these fusion proteins were successfully expressed. Under laser confocal microscope, GFP fused with fragment T4 （aa71-128） localized mainly in the nucleolus; GFP fused with fragment T6 （aa71-380） localized diffusely in the nucleus; while other fusion proteins with TMSG-1 （aal-380） or fragment T1 （aal-70） , T2 （aal-128） , T3 （aal29-380） and T5 （aa71-179） localized in the cytoplasm. Fragment T4＆l19q28 was generated from T4 with deletion of 10 amino acid of the C terminal. GFP fused with fragment T4a119-I28 remained in the nucleus, but no longer in the nucleolus. Conclusions There is a nucleolar localization signal （aa119-128 RRRRNQDRPS） within TMSG-1. This finding may have laid the foundation for further investigations into subcellular localization and function of TMSG-1.