核因子NF-κB p65亚基TAD的克隆及表达

Clone and expression of nuclear factor κB p65 TAD

目的获得NF-κB p65亚基转录激活区域(transcription activation dom ain,TAD),构建真核表达载体并在内皮细胞中检测其表达。方法培养人脐静脉内皮细胞,提取总RNA,经RT-PCR得到p65 TAD基因片段,测序正确后插入带有绿色荧光蛋白的真核表达载体pEGFP-N1,构建表达载体pEGFP-N1-p65 TAD,然后转染内皮细胞,荧光显微镜下观察表达情况,同时用W estern b lot分析蛋白的表达。结果有效地扩增了p65 TAD 783 bp的目的基因片段。酶切结果表明所构建的含p65 TAD基因的质粒与设计相同。p65 TAD在脐静脉内皮细胞中得到了高效的表达。结论成功地构建了p65 TAD的真核表达载体并实现了在内皮细胞中的高效表达。

Objective To acquire NF-κB p65 TAD （ transcription activation domain） and construct its eukaryotic expression vector and express it in endothelial cells, Methods Human umbilical vein endothelial cells （HUVECs） were cultured and total RNA was extracted. The p65 TAD gene was amplified by RT-PCR. After sequencing, the p65 TAD gene was inserted into the eukaryotic expression vector pEGFP-N1 with the green fluorescence protein, named pEGFP-N1-p65 TAD, pEGFP-N1-p65 TAD was transfected into HUVECs and its expression was observed under fluorescence microscope and analyzed by Western blotting. Results p65 TAD （288 -548 ） was cloned successfully. The constructed plasmid including p65 TAD gene was identical to the designed, p65 TAD gene was expressed successfully in HUVECs. Conclusion The construction of eukaryotic expression vector including p65 TAD gene and its expression in HUVECs are very successful,