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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

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

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【作者】 郭建新许予明赵国强张瑞宋波张世杰张世峰李京红

【Author】 GUO Jian-xin~*,XU Yu-ming,ZHAO Guo-qiang,ZHANG Rui,SONG Bo,ZHANG Shi-jie,ZHANG Shi-feng,LI Jing-hong.~*Department of Neurology,the First Affiliated Hospital,Zhengzhou University,Zhengzhou 450052,China

【机构】 郑州大学第一附属医院神经内科郑州大学基础医学院微生物与免疫教研室郑州大学第一附属医院神经内科 郑州450052郑州450052

【摘要】 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。

【Abstract】 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.

【关键词】 SOX1真核表达载体
【Key words】 SOX1eukaryotic expression vector
【基金】 国家自然科学基金资助项目(30570624);河南省教委杰出人才资助项目;郑州市重大科技攻关项目(03BA02BMYB05)
  • 【文献出处】 神经损伤与功能重建 ,Neural Injury and Functional Reconstruction , 编辑部邮箱 ,2006年01期
  • 【分类号】R346
  • 【下载频次】121
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