【摘要】 目的构建蛋白质精氨酸甲基转移酶2(PRMT2)及其差异剪接体与绿色荧光蛋白(GFP)的真核表达载体,转染后观察其融合蛋白在乳腺癌MCF-7细胞中的表达及亚细胞定位,为进一步研究PRMT2基因及其新的差异剪接体在乳腺癌中的作用奠定基础。方法以p GEM-T-PRMT2/α/β/γ载体为模板,设计引物,PCR扩增目的基因,并将PCR产物克隆至pc DNA3.1/NT-GFP-topo载体,转化后将阳性克隆扩增,对PCR产物进行跑胶鉴定和测序。提取pc DNA3.1/NT-GFP-PRMT2/α/β/γ及空载体pc DNA3.1/NT-GFP质粒,用脂质体介导转染MCF-7细胞,在激光共聚焦显微镜下,观察外源性PRMT2/α/β/γ融合蛋白在MCF-7细胞中的亚细胞定位。采用Western blot法检测各重组融合蛋白在MCF-7细胞中的表达。结果各GFP在细胞中均有表达,但其在细胞中的分布位置不一致。PRMT2α与PRMT2γ的融合蛋白同PRMT2的分布一致,均聚集于核仁外的核浆,胞质中有少许分布;而PRMT2β和空载体的荧光蛋白的分布一致,都均匀分布于细胞的胞质和胞核,包括核仁部分。Western blot法检测表明各重组融合蛋白在MCF-7细胞中均有表达。结论PRMT2及其各剪接体的亚细胞定位不同,可能提示其在功能方面存在差异。更多还原

【Abstract】 Objective To construct eukaryotic expression vectors pc DNA3. 1 / NT-GFP-PRMT2 and its splicings pc DNA3. 1 / NT-GFP-PRMT2α / β / γ and transfected into breast cancer MCF-7 cells,observe the subcellular localization of GFP-fusion protein in order to investigate the role of PRMT2 and its splicings in breast cancer. Methods The PRMT2 and its splicings genes were amplified from the vectors p GEM-T-PRMT2 / α / β / γ,then subcloned into pc DNA3. 1 / NT-GFP-topo vector and sequenced. The recombinant pc DNA3. 1 / NT-GFP-PRMT2 / α / β / γ,were transfected into breast cancer MCF-7 cells by lipofectamine respectively. The expression of green fluorescent protein was observed under laser scanning microscope,and the expression levels of PRMT2α / β / γ fusion protein were indentified by western blot. Results The expression of GFP fusion protein of PRMT2 and its splicings were observed under the laser scanning microscope,indicating that PRMT2α and PRMT2γ GFP fusion protein resulted in the nuclei except the nucleolus,cytoplasm has diffusion to scatter,as well as that of PRMT2. PRMT2β and N-GFP protein showed scatter homogeneously in nuclei and cytoplasm. Western blot indicated the recombinant PRMT2 and its splicings PRMT2α / β / γ genes were respectively expressed in breast cancer MCF-7 cells. Conclusion The subcellular localization of PRMT2 and its splicings are different and which maybe suggests they that the distinct roles in breast cancer.更多还原