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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。
Objective To observe the expression and localization of Myc tagged ERβ1293 recombinant eukaryotic expression vector in HEK293T cells. Methods The eukaryotic expression vector pEGFP-N1-ERβ1293 was used as a template, and the full-length coding region of ERβ1293 gene was amplified by PCR. Myc-tagged ERβ1293 recombinant eukaryotic vector was transiently transfected into HEK293T cells. The expression and localization of pcDNA3.1-Myc-ERβ1293 in HEK293T cells was identified by Western blotting and indirect immunofluorescence (IF). Results The pcDNA3.1-Myc-ERβ1293 recombinant eukaryotic expression vector was successfully constructed and transfected into HEK293T cells. The protein bands were detected by Western blotting. The transfected cells after IF treatment were observed under the confocal microscope, and the fluorescence was localized in the cytoplasm. CONCLUSION: The ERβ1293 coding region of ERβ isoforms was deleted in the previous experiment and the fourth exon was deleted, which led to its weakening or disappearance of the ligand binding ability. Therefore, the localization of the encoded protein in the target gene was changed in the cells.