小鼠LIF基因分泌型真核表达载体的构建及其应用研究

目的构建小鼠白血病抑制因子的真核表达载体,有助于开展高其他实验动物的胚胎干细胞的定向分化研究。方法运用RT-PCR技术,克隆含信号肽和不含信号肽的小鼠分泌型白血病抑制因子,通过pMD18-Tsimple载体和pBS-T载体过渡,酶切进行初步鉴定。结果分别经过酶切鉴定,成功构建了小鼠LIF基因真核表达载体pSecTag-LIF(sp )和pSecTag-LIF(sp-)。结论构建小鼠白血病抑制因子的真核表达载体有效可行,不仅为进一步研究LIF细胞因子所诱导的维持胚胎干细胞未分化状态的分子机制奠定了基础,而且为各种转基因实验动物的研究提供了一种新的方法。

Construction and Application of Eukaryotic Expression Vector of Mouse LIF

Objective Constructing the eukaryotic expression vector of mouse LIF and conducing to improve the directional differentiation research of ES cell about other laboratory animals. Method Mouse secreted LIF with and without signal peptide were cloned from mouse livers by RT-PCR, and then subcloned into pBS-T vector and pMD18-T simple vector. Digesting fragments were recovered and inserted into pSecTag/Hygro with molecular cloning technique, and digested analysis by restrictive enzymes. Results By digestion identification with restrictive enzymes the LIF gene was cloned into eukaryotic expression vector pSecTag/Hygro successfully and the expression vectors we constructed were called pSecTag-lif(sp ) and pSecTag-lif(sp-). Conclusion It is valid to construct the eukaryotic expression vector of mouse LIF, and this not only establishs the base of molecule principle that maintains the undifferentiated ES cell, but also can provide a new method for the research of transgenic laboratory animals.