摘要
目的:探讨HOXA11基因顺式作用元件是否存在启动子及孕激素应答元件序列。方法:采用PCR技术以人基因组DNA为模板,扩增含HOXA11顺式作用元件序列的DNA片段;将扩增片段与无启动子的强化绿色荧光蛋白报告基因载体pEGFP-1多克隆位点连接,构建HOXA11基因顺式作用元件的表达载体pEGFP-HOXA11;用脂质体法将表达载体转染到Ishikawa细胞中,在荧光显微镜下观察重组HOXA11基因顺式作用元件的启动活性,以及转染细胞经孕酮处理后细胞内绿色荧光蛋白表达水平的变化。结果:成功地将HOXA11基因顺式作用元件克隆到报告基因载体pEGFP-1中,酶切和PCR鉴定以及DNA序列分析无误;转染pEGFP-HOXA11表达载体的Ishikawa细胞能表达发出绿色荧光的报告基因蛋白,而且经孕酮处理后绿色荧光强度明显增强。结论:HOXA11基因顺式作用元件具有启动子活性和孕激素应答元件功能。
Objective： To explore the promoter position and existence of progesterone response element in HOXA11 gene cis-acting element. Methods： The cis-acting element DNA segment of HOXA11 gene was synthesized through polymerase chain reaction （PCR） from genomic DNA of human deciduas and was cloned into the multiple cloning site of promoterless enhanced green fluoresce protein （EGFP） vector pEGFP- 1. The recombinant expression plasmid pEGFP-HOXA11 was then transfected into Ishikawa cells by lipofectamine methods. By fluorescent microsocope, HOXA11 gene promoter activity and GFP expression levels were observed in Ishikawa cells transfected by pEGFP-HOXA11 with treatment of progesterone. Results： The length, position and orientation of inserted DNA segment containing cisction element of HOXA11 gene in recombinant expression plasmid pEGFP-HOXA11 were demonstrated to be correct by means of restriction digestion, PCR and DNA sequence analysis. The green fluorescence could be detected in Ishikawa cells transfected by recombinant expression plasmid pEGFP-HOXA11, and the more intensive green fluorescence was observed in the cells treated with progesterone after transfenction of pEGFPHOXA11. Conclusion： The cis-acting element of HOXA11 gene has promoter activity and progesterone response element effect.
出处
《生殖与避孕》
CAS
CSCD
北大核心
2008年第11期641-645,共5页
Reproduction and Contraception
基金
国家自然科学基金资助项目,基金项目号:30471814
