摘要
目的构建E381的绿色荧光蛋白融合表达质粒pEGFP-N1／E381和pEGFP—C1／E381，并观察E381在MCF-7乳腺癌细胞的核定位情况。方法用PCR方法扩增E3B1cDNA，构建绿色荧光蛋白融合表达质粒pEGFP-N1／E381和pEGFP—C1／E381，酶切、测序鉴定后，瞬时转染MCF-7乳腺癌细胞株，用荧光显微镜观察E381的核定位情况。结果酶切结果与序列测定说明获得pEGFP-N1／E381和pEGFP-C1／E381重组质粒，转染分析可见E381可定位于MCF-7乳腺癌细胞核内。结论成功构建了E381的绿色荧光蛋白融舍表达质粒pEGFP-N1／E381和pEGFP—C1／E381，在MCF-7乳腺癌细胞株中，E381除存在于胞浆外，也可定位于细胞核内，可能与其参与核内基因的转录调控有关。
Objective To construct the recombinant plasmid pEGFP-N1/E3B 1 and pEGFP-C1/E3B 1 expressing the fusion green fluorescent protein E3B1-EGFP and pEGFP- E3B1, and observe the nuclear localization of E3B1 in MCF-7 cells. Methods A cDNA fragment encoding for E3B1 was amplified by PCR and inserted into MCS of green fluorescent protein pEGFP-N1 and pEGFP-C1. After identification of the positive clone by digestion of restriction endonuclease and sequencing, the recombinant construct was transiently transfected into MCF-7 cells and the nuclear localization of the fusion protein E3BI-EGFP and EGFP-E3B1 was observed by fluorescence microscope. Results The recombinant plasmid pEGFP-N1/E3B1 and pEGFP-C1/E3B1 which fusion protein expressed by the plasmid has a correct reading frame, can be localized in nuclei of MCF-7 cells. Conclusion the recombinant plasmid pEGFP-N1/E3B1 and pEGFP-C1/E3B1, which encodes fusion protein of E3B1-EGFP and pEGFP- E3BI, was successfully constructed. E3B1 was demonstrated to be localized in nuclei of MCF-7 ceils except as a cytosolic protein.
关键词
E381
MCF-7
乳腺癌细胞
核定位
EPS8-SH3-domain binding protein-1
MCF-7 cells
Nuclear localization
