摘要
目的阐明核糖核酸(RNA)结合蛋白T细胞内抗原相关蛋白(TIAR)的结构域与细胞内定位之间的关系。方法细胞免疫荧光检测SMCC-7721细胞内源性TIAR的细胞内分布,亚克隆构建TIAR全长及缺失体的增强型绿色荧光蛋白(EGFP)表达质粒,脂质体法瞬时转染SMCC-7721细胞,在荧光显微镜下观察融合蛋白在细胞内的荧光分布情况,免疫印迹检测融合蛋白的表达情况。结果细胞免疫荧光结果显示在SMCC-7721细胞中TIAR主要浓集于细胞核,细胞浆也有少量弥散分布。酶切鉴定及测序结果显示TIAR全长及缺失体的pEGFP-C3重组表达质粒构建成功。EGFP-TIARFL(TIAR全长)主要分布于细胞核;而EGFP-TIARRRM123(TIAR的N端3个RNA结合结构域)弥散分布于整个细胞;EGFP-TIARQ-rich(TIAR的C端谷胺酰胺富含结构域)浓集于细胞核,但浓集程度要比TIARFL-EGFP弱。免疫印迹证实过表达的TIAR全长及缺失体的EGFP融合蛋白分子量大小符合预期。结论我们的结果提示C端谷氨酸富含结构域参与TIAR细胞内定位的调节。
Purpose To explore the relationship between structure and subcellular localization of an RNA-binding protein, TIAR. Methods Immunofluorescence was used to detect the subcellular localization of endogenous TIAR. Subcloning technique was performed to construct the expression plasmids of EGFP-fused TIAR full length and truncated mutants. The subcellular localization of TIAR full length and truncated mutants were observed by using fluorescent microscopy. Western blotting was used to confirm the expression of EGFP-fused proteins. Results Endogenous TIAR was primarily concentrated in the nucleus of SMCC-7721 cells. Electrophoresis of digested plasmids and Sequencing confirmed that we constructed the recombinant plasmids successfully. EGFP-TIAR^FL was mainly concentrated in the nucleus. EGFP-TIAR^RRM123 was localized both in nucleus and cytoplasm. EGFP-TIAR^Q-rich was concentrated in the nucleus, but not as significant as EGFP-TIAR^FL. Further more, Western blotting demonstrated that the recombinant proteins were successfully expressed in SMCC-7721 cells in the predicted molecular weight. Conclusions Our results indicated that C-erminal Q-rich domain was involved in the regulation of subcellular localization of TIAR.
出处
《复旦学报（医学版）》
CAS
CSCD
北大核心
2006年第2期229-232,共4页
Fudan University Journal of Medical Sciences
基金
国家自然科学基金(30070412)
复旦大学青年教师教学与科研启动基金(CHF101005)资助
关键词
T细胞内抗原相关蛋白
细胞内定位
增强型绿色荧光蛋白
T-cell-restricted intracellutar antigen-related protein
subcellular localization
enhanced green fluorescence protein
