小鼠SRY同源盒基因SOX1的克隆及其真核表达载体pEGFP-C3-SOX1的构建

目的:克隆小鼠SRY同源盒基因SOX1的全长cDNA,构建其携带增强型荧光蛋白的真核表达载体pEGFP-C3-SOX1,为进一步研究SOX1在间充质干细胞神经分化中的作用奠定基础。方法:采用RT-PCR方法从小鼠早期胚胎中扩增带有EcoRⅠ和HindⅢ双酶切位点的SOX1 cDNA片断,与pGEM-T载体连接后转化感受态细菌JM109,然后通过蓝白筛选、酶切技术筛选、鉴定出阳性菌落并进行DNA测序。用EcoRⅠ和HindⅢ双酶切获得目的片断,再与EcoRⅠ和HindⅢ双酶切过的pEGFP-C3质粒相连接,重组子转化JM109感受态大肠杆菌,酶切方法鉴定阳性菌落。结果:多酶切及DNA测序结果表明提取及纯化的pEGFP-C3-SOX1质粒含有SOX1 cDNA碱基片段,方向及大小正确。结论:从小鼠胚胎中可成功克隆SOX1全长cDNA,并构建其真核表达载体pEGFP-C3-SOX1。

Cloning of Mouse SRY-Box Gene SOX1 and Constructing of Eukaryotic Expression Vector pEGFP-C3-SOX1

Objective:To clone the entire coding sequence and construct a mouse sox1 expression vector PEGFP-C3-sox1 for studying the effect of SOX1 on neural differentiation of stem cells.Methods: The fragment of sox1 containing EcoRI and HindIII was amplified by RT-PCR from the mouse earlier embryo and then cloned into pGEM-T Easy vector.The recombinant plasmid was identified by restriction-endonuclease digestion and DNA sequencing.The fragment of sox1 was digested by EcoRI and HindIII and subclonded into the expression vector pEGFP-C3,which was denominated with pEGFP-C3-SOX1,and then the recombinant plasmid was identified by restriction-endonuclease double digestion.Results: Restriction-endonuclease digestion and DNA sequencing showed that the recombinant plasmid pEGFP-C3 contained the fragment of sox1.Conclusion:The entire coding sequence of mouse SRY-Box gene SOX1 was successfully cloned and sox1 expression vector pEGFP-C3-sox1 was constructed.