OSP-1启动子在猪卵巢颗粒细胞中的转录活性研究

Transcriptional Activity of Ovarian-Specific Promoter 1 in Porcine Granulosa Cells

为获得能在猪卵巢颗粒细胞中高效表达的启动子,本文检测了大鼠卵巢特异性启动子OSP-1(Ovarian specific promoter)在猪卵巢颗粒细胞中的转录活性。参考大鼠OSP-1启动子序列扩增大鼠OSP-1片段,将其定向克隆到pc DNA3.1(+)中替代CMV启动子,并在其下游插入Zs GREEN基因片段,构建真核表达载体p OSP-1-Zs GREEN;重组质粒在脂质体LipofectamineTM2000的介导下转染不同细胞,在荧光显微镜下观察Zs GREEN表达情况,Realtime-RT-PCR方法检测该启动子在不同细胞中启动Zs GREEN基因表达的活性。结果表明:经克隆测序,得到了465bp OSP-1启动子序列,重组质粒转染卵巢颗粒细胞、Hela细胞可以观察到绿色荧光。Realtime-RT-PCR结果显示:Zs GREEN基因在卵巢颗粒细胞、Hela细胞中高表达,在肌细胞中表达量极低;说明OSP-1启动子可驱动外源基因在猪卵巢颗粒细胞中高效表达,可为构建可用于猪卵巢颗粒细胞的表达载体提供了启动子。

To obtain an efficient promoter in porcine granulosa cell,rat ovarian specific promoter OSP- 1 was cloned and its transcriptional activities in several kind of cells were detected. OSP- 1 promoter was amplified according to rat OSP- 1 sequence and inserted into the vector pc DNA3.1(+)to replace CMV promoter,following with Zs Green sequence as a reporter.The recombinant plasmid was transfected into porcine granulosa cells,Hela cells and myocytes;the fluorescence in cells were observed under microscope,the expression of Zs Green m RNA in different cells were detected by realtime- PCR,the results showed that the Zs Green gene was high expressed in ovarian granulosa cells and Hela cells,very low expressed in myocytes,which indicated that OSP- 1 promoter could promote the expression of exogenous gene efficiently in porcine granulosa cells.