缺失激素结合区的人糖皮质激素受体突变体的研究

Investigations of glucocorticoid receptor mutant without ligand binding domain

目的:构建缺失激素结合区(LBD)的人糖皮质激素受体突变体表达载体,并研究其表达和功能。方法:运用RT-nested PCR扩增人糖皮质激素受体(GR)基因不含编码LBD的cDNA序列,将PCR产物定向克隆至pEGFP-N2质粒载体,测序筛选编码正确的重组质粒;荧光显微镜观察GR突变体重组质粒转染人巨噬细胞株U937后的表达情况;相对荧光素酶法检测转染后细胞GR转录激活活性。结果:扩增出长1601 bp的cDNA序列,测序证实:克隆片段与Genebank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GR转录激活活性增强。结论:成功构建缺失LBD的GR突变体表达载体,该突变体具有不依赖激素的GR转录激活活性。

Objectlve：To construct expression vector of human glucocorticoid receptor mutant. Methods ：cDNA sequence of human glucocorticoid receptor mutant having no LBD was amplificated through RT-nested PCR, PCR product was directionaily cloned to pEGFP-N2 plasmid vector, and recombinant plasmids were screened through sequencing. Expressions of recombinant plasmids in human macrophage cell line U937 were observed through fluorescence microscope. Transcriptional activation activity of vailant was evaluated through was obtained, and result methods of relative activity of luciferase. Results ：The 1601 bp length cDNA of sequencing confirmed the cloned cDNA sequence is completely coincidence with that of glucocorticoid receptor in Genebank. Expression products of recombinant plasmid mainly located in nucleus. Transcriptional activation activity incresed after recombinant plasmid transfected. Conclusion：Expression vector of glucocorticoid receptor mutant was constructed successfully, which has transcriptional activation activity independent of glucocorticoid.