繁殖候选基因Lhx8的真核表达和定位分析

The eukaryotic expression and localization analysis of reproductive candidate gene Lhx8

从猪卵巢组织中提取总RNA为模板,采用RT-PCR方法获得了猪Lhx8基因的完整开放阅读框,将该基因克隆到真核表达载体pEGFP-N1中,经酶切分析和测序证明成功构建了真核重组质粒pEGFP-N1-Lhx8。将成功构建的pEGFP-N1-Lhx8分别转染猪肾细胞PK-15和猪颗粒细胞(GCs),转染后48 h,通过荧光显微镜观察对猪Lhx8基因在这2种细胞中的定位特点进行分析。结果显示:绿色荧光呈点状分布,根据绿色荧光分布的位置,推测Lhx8蛋白很可能位于细胞核中。

The total RNA extracted from a porcine ovary was used as a templet,and the full open reading frame（ORF）of porcine Lhx8 gene was obtained by RT-PCR. The full ORF of Lhx8 was cloned into pEGFP-N1 vector, and the recombinant plasmid pEGFP-N1-Lhx8 was constructed and confirmed by restriction analysis and sequencing. The recombinant plasmid was transfected into PK-15 cells and porcine granulosa cells（GCs） respectively. At 48 h after transfection, the localization of Lhx8 protein in the two kinds of cells was analyzed by fluorescent microscope. The results showed that the green fluorescence assumed spot distribution. According to the location of the green fluorescence, the Lhx8 protein was probably in the nucleus.