比格犬ERβ_(1293)基因真核载体的构建及其在HEK293T细胞中的表达和定位

Expression and localization of eukaryotic expression vectors of Beagles ERβ_(1293) gene in the HEK293T cells

目的观察比格犬Myc标签ERβ1293重组真核表达载体在HEK293T细胞中的表达及定位。方法以pEGFP-N1-ERβ1293重组真核表达载体为模板,PCR保真酶扩增得到ERβ1293基因编码区全长。Myc标签ERβ1293重组真核载体瞬时转染HEK293T细胞,运用蛋白质印迹技术(Western blotting)和间接免疫荧光技术(indirect immunofluorescence,IF)鉴定pcDNA3.1-Myc-ERβ1293在HEK293T细胞中的表达和定位情况。结果成功构建pcDNA3.1-Myc-ERβ1293重组真核表达载体,转染至HEK293T细胞中。Western blotting检测有蛋白条带表达,共聚焦显微镜下观察IF处理后的转染细胞,荧光定位于细胞质。结论前期实验得到比格犬ERβ剪切异构体ERβ1293编码区序列,缺失第四外显子,导致其与配体结合能力减弱或消失,因此目的基因编码蛋白定位在细胞中发生变化。

[ Abstract] Objective To construct recombinant eukaryotic expression vectors of Beagles ERβ1293 , and identify the expression and localization of ERβ1293 in the human embryonic renal HEK293T cells by Western blotting and indirect immunofluorecent assay. Methods The ERβ129s gene was amplified by PCR using pEGFP-N1-ERβ1293 as a template. The recombinant eukaryotic expression vector of pcDNA3. 1-Myc-ERβ1293 was constructed, and transfected it into HEK293T cells. The expression of pcDNA3.1-Myc-ERβ1293 was detected by Western blot, and its localization was detected by indirect immunofluorecence assay. Results The expression of pcDNA3. 1-Myc-ERβ1293 was constructed, and successfully identified in the HEK293T cells using Western blotting, and detected only in the cytoplasm using IF by laser scanning confocal microscopy. Conclusions In the previous experiment, the coding sequence of.Beagles ERβ1593 splice variants has been obtained, which is lack of exon 4, and its ligand binding ability is reduced or disappeared, so that the intracellular localization of ERβ1293 codeing protein is changed.