肿瘤转移相关基因TMSG-1编码蛋白的定位

Protein localization of a tumor metastasis associated gene TMSG-1

目的 明确肿瘤转移相关基因TMSG 1编码蛋白在细胞内的定位。方法 计算机分析TMSG 1的跨膜区 ,构建GFP与TMSG 1融合蛋白表达载体pEGFP C1 TMSG 1,以LipofectAMINE介导转染人前列腺癌细胞PC 3M 1E8,荧光显微镜与共聚焦显微镜下观察。结果 计算机分析表明所示 ,TMSG 1有四个跨膜区 (aa33～ 4 8,aa6 4～ 79,aa114～ 130 ,aa15 8～ 174 ) ,推测其为跨膜蛋白。PSORTⅡ软件分析TMSG 1蛋白定位于胞浆 (可靠性 94 .1% ) ,具体位置为内质网 6 6 .7%、线粒体 11.1%、质膜 11.1%、高尔基体 11.1%。显微镜下观察TMSG 1 GFP融合蛋白表达于胞浆 ,呈不均匀的颗粒状、环状分布 ;而对照的GFP蛋白则在胞核和胞浆呈弥漫性分布。结论 TMSG 1蛋白定位于细胞内膜系统 ;利用GFP进行的亚细胞定位具有精确、快速、可重复等优点 ,是研究基因与蛋白定位的有效方法。

Objective To determine the subcellular localization of the encoded protein of tumor metastasis associated gene TMSG 1. Methods Transmembrane domain was analyzed by using computer. Fusion protein expression vector pEGFP C1 TMSG 1 was constructed and transfected into prostate cancer cell line(PC 3M 1E8). Results were observed by using fluorescence microscope and laser confocal microscope. Results Computer analysis demonstrated that TMSG 1 had four transmembrane domains (aa33～48, aa64～79, aa114～130, aa158～174)and TMSG 1 was supposed to be a transmembrane protein. PSORTⅡsoftware analysis predicted TMSG 1 protein was located in cytoplasm (Reliability: 94.1%). The definite location was endoplasmic reticulum 66.7 %, mitochondria 1.1 %, plasma membrane 11.1 % and Golgiosome 11.1 %. Microscope observation showed that TMSG 1 GFP fusion protein was expressed in cytoplasm and took on granular and annular distribution, while GFP protein was expressed in nucleolus and cytoplasm, and had permeating distribution. Conclusion TMSG 1 located in the intracellular membrane system. The subcellular localization of GFP had such advantages as precision, efficiency and convenience. It is a kind of powerful tool in gene research and protein localization.