人糖皮质激素受体变异体cDNA序列克隆和表达及其活性研究

cDNA cloning and expression of human glucocorticoid receptor variant

目的构建人糖皮质激素受体变异体表达载体,研究其表达和功能。方法运用RT-nested PCR扩增糖皮质激素受体不含有编码LBD(ligand b ind ing dom ain)的cDNA序列,将PCR产物定向克隆至pEGFP-N2质粒载体,测序筛选重组质粒;荧光显微镜观察重组质粒转染表达情况;相对荧光素酶法检测GR转录激活活性。结果扩增出长1 601 bp的cDNA序列,测序证实:克隆片段与Genbank该基因序列同源性为100%;重组质粒表达产物定位于细胞核;转染组细胞转录激活活性增强。结论成功构建糖皮质激素受体变异体的表达载体,该变异体具有不依赖激素的转录激活活性。

Objective To construct the expression vector of human glucocorticoid receptor variant and investigate its expression and function. Methods cDNA sequence of human glucocorticoid receptor variant without LBD was amplificated by RT-nested PCR, directionally cloned to pEGFP-N2 plasmid vector, and screened through sequencing. The expressions of recombinant plasmids were observed through fluorescence microscope. The transcriptional activation activity of variant was evaluated by the relative activity of luciferase. Results The cDNA of 1 601 bp was obtained, and the sequencing confirmed the cloned cDNA sequence was completely coincident with that of glucocorticoid receptor in GenBank. Expression products of recombinant plasmid mainly located in nucleus. The transcriptional activation activity increased after recombinant plasmid transfection. Conclusion Expression vector of glucocorticoid receptor variant was constructed successfully, which has transcriptional activation activity independent of glucocorticoid.