猪白血病抑制因子的真核表达与活性分析

Eukaryotic Expression of Porcine LIF and the Assay of Its Bioactivities

采用分子克隆技术构建猪白血病抑制因子(LIF)的真核表达载体,表达重组猪LIF蛋白并分析其活性。酶切和DNA测序显示,所构建的真核表达质粒pcDNA3.1-pLIF-MycHis含有编码分泌型猪LIF蛋白的cD-NA序列;免疫印迹分析显示,分泌到上清中的重组蛋白在相对分子质量约37 000的位置上有明显的增强表达条带;活性分析结果显示,重组蛋白能够维持小鼠E-14胚胎干细胞在未分化状态。该结果显示重组猪LIF蛋白在CHO细胞中进行了正确表达并具有很好的生物活性,能够用来培养小鼠胚胎干细胞,为用于猪胚胎干细胞系的研究奠定了良好的基础。

The expression plasmid of porcine LIF in the eukaryotic system was constructed by moleoular cloning technique, the recombinant porcine LIF protein was expressed exactly, and the bioactivity of recombinant porcine LIF protein was further assayed immediately. The results indicated that the constructed expression plasmid pcDNA3.1-pLIF-MycHis was identified by DNA sequencing and restriction enzyme digestion; the expression of the recombinant porcine LIF protein was performed exactly in CHO cell; in the conditioned media, there is a single glycosylated band at 37 kD visualized by immunoblotting; the recombinant porcine LIF expressed exactly in CHO cell was able to maintain mouse E-14 ES cells in undifferentiated status. As a result, the recombinant porcine LIF with bioactivity expressed exactly in ClIO cell can be used to culture mouse ES cell, and it will be useful in establishing stable pluripotent porcine ES cells for further manipulation.