摘要
目的克隆大鼠抑制素α亚基(Inhα)启动子,建立报告基因表达系统,初步分析其在睾丸支持细胞中的转录活性。方法采用降落聚合酶链反应(touch-down PCR)法,从大鼠睾丸组织DNA中扩增三段不同长度的Inhα启动子序列(395、606、775 bp),克隆入载体pEGFP-N1,构建绿色荧光蛋白的报告基因系统,转染大鼠睾丸支持细胞,荧光显微镜观察绿色荧光表达情况。结果成功构建带有Inhα启动子的绿色荧光蛋白报告基因表达系统。三段重组质粒转染支持细胞后均可见绿色荧光表达,但荧光量和强度均弱于CMV启动子,其中395片段最为显著。结论 Inhα启动子在睾丸支持细胞中的转录活性弱于CMV启动子,可能与启动子本身的序列结构有关。
Objective： To clone rat inhibin alpha gene （Inha） promoter and analyze its transcriptional activity with reporter gene expression system in sertoli cells. Methods： Three different sequences of Inha promoter （395, 606, 775 bp） from rat testicle DNA were amplified with touch-down PCR and cloned with pEGFP-N1 to construct reporter gene expression system. And then the 3 recombined plasmids were transfected into rat sertoli cells to examine the expression of EGFP by use of fluorescent microscopy. Results： The EGFP reporter gene expression vector with Inha promoter sequences were constructed successfully. The expression of green fluorescence observed in sertoli cells transfected with either of 3 recombined plasmids was fewer and weaker than that with CMV promoter （positive control）, especially for sequences 395. Conclusions： The transcriptional activity of Inhα promoter was less than that of CMV promoter in sertoli cells, which could be due to the sequence character of Inha promoter.
出处
《生殖医学杂志》
CAS
2010年第5期429-433,共5页
Journal of Reproductive Medicine
基金
国家自然科学基金资助项目(No.30770814)
