大鼠糖皮质激素受体变异体cDNA序列克隆和表达及功能研究  

Investigations of cloning cDNA sequence and expression and function of human glucocorticoid receptor variant

作　　者：张芳[1] 钱桂生[1] 钱频[1] 陈龙[2] 李树钧[1] 陈维中[1] 

机构地区：[1]第三军医大学新桥医院全军呼吸研究所,重庆400037 [2]兰州军区兰州总医院

出　　处：《西北国防医学杂志》2005年第1期1-3,F003,共4页Medical Journal of National Defending Forces in Northwest China

基　　金：国家自然科学基金资助项目 (3 9970 3 2 9)

摘　　要：目的 :构建大鼠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 -nestedPCR扩增糖皮质激素受体不含有编码LBD结构域的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后的表达情况 ;相对荧光素酶法检测转录激活活性。结果 :扩增出长 16 12bp的cDNA序列 ,测序证实 :克隆片段与Genebank该基因序列同源性为 10 0 % ;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 rat glucocorticoid receptor variant, investigate its expression and function. Methods: cDNA sequence of rat glucocorticoid receptor variant having no LBD was amplificated through RT-nested PCR, PCR product was directionally cloned to pEGFP-N2 plasmid vector, and recombinant plasmids were screened through sequencing; Expressions of recombinant plasmids were observed through fluorescence microscope; Transcriptional activation activity of variant was evaluated through methods of relative activity of luciferase. Results: 1 612 bp length cDNA sequence was obtained, and result of sequencing confirmed the cloned cDNA sequence is completely coincident with sequence 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 variant was constructed successfully, which has transcriptional activation activity independent of glucocorticoid.

关 键 词：糖皮质激素受体 LBD 转录激活活性 

分 类 号：R346[医药卫生—基础医学]