【摘要】 目的构建hERα真核表达载体并证实融合蛋白在细胞内表达及定位。方法以pcDNA3.1-flag-hERα重组质粒(Dr.Muyan M馈赠)为模板,PCR扩增hERα全长编码基因,亚克隆至pEGFP-C1表达载体。将构建的重组质粒测序并转染到乳腺癌细胞MCF-7中,提取细胞蛋白进行Western blot检测。利用共聚焦激光扫描显微镜观察pEGFP-hERα在MCF-7细胞内定位。结果 hERα全长基因序列克隆到了真核表达载体pEGFP-C1中,酶切鉴定片段为1800bp。Western blot检测到了融合蛋白表达,分子量约为95KD,pEGFP-hERα在乳腺癌细胞MCF-7细胞内定位于细胞核内。结论成功的构建了hERα全长基因真核表达载体pEGFP-hERα,融合蛋白定位于乳腺癌细胞核内。更多还原

【Abstract】 Objective To construct recombinant expression plasmid of human estrogen recptor α(hERα) gene and identify its protein expression and localization. Methods The hERα coding sequence was amplified by polymerase chain reaction(PCR) method and subcloned into pEGFP-C1 vector. After the target region was sequenced,the plasmid was transfected into MCF-7 cell lines. The expression of the recombinant plasmid in breast cancer cells(MCF-7 cells) was proved by Western blot and the localization of pEGFP-hERα C was observed by using laser scanning confocal microscopy. Results HERαwas constructed into expressing vector pEGFP-C1 successfully,the length of the fragment was 1800bp identified by restriction enzymes digestion. The expression of pEGFP-hERα fusion protein was detected by Western blot in MCF-7 cells,with a molecular weight 95KD,and localized in the nucleus of MCF-7 cells. Conclusion The recombinant plasmid of pEGFP-hERαwas constructed successfully and the fusion protein was localized in nucleus of MCF-7 cells.更多还原