pEGFP-PDX-1真核表达载体的构建

Construction of mammalian expressing vector pEGFP-PDX-1

  • 摘要: 目的: 构建真核表达载体pEGFP-PDX-1,为基因水平研究PDX-1在肝干细胞定向分化中的调控机制提供理论参考。方法: 参考分子克隆技术,采用PCR的方法从SK900/BLSCR IPT质粒中扩增PDX-1,同时将一个单个限制性酶切位点连接反应转变为以H indⅢ和BamHⅠ为末端的双限制性内切酶位点连接反应,将PDX-1 cDNA定向插入pEGFP-C1的多克隆位点中,构建重组表达质粒pEGFP-C1-PDX-1,并对重组子进行Sm aⅠ单个限制性内切酶酶切鉴定。结果: 构建了含有PDX-1 cDNA的真核表达载体pEGFP-PDX-1。结论: 将PDX-1插入pEGFP-C1的C-末端,提高PDX-1在真核细胞中的表达,而且不影响其目的蛋白的结构和功能,对研究PDX-1调控肝干细胞的定向分化机制具有重要的意义。

     

    Abstract: Objective: To study the induction mechanism of PDX-1,the key gene to regulate the development of pancreatic endocrine cells,during the differentiation of hepatic stem cells into insulin-producing cells,we try to construct a mammalian-expressing vector carrying PDX-1 to search for a proper way to induce the differentiation of hepatic stem cells into islet β-cells from genic level.Methods: All experiment methods are performed according to the standard protocols of molecular clone technology.Polymerase chain reaction(PCR) was used to proliferate PDX-1 cDNA from SK900/BLSCRIPT plasmid,producing an 858bp band with HindⅢ and BamHⅠat both sides of the DNA.Cloned PDX-1 cDNA into the MCS of pEGFP-C1 to get a recombined mammalian expressing plasmid pEGFP-C1-PDX-1,then SmaⅠdigested and separated by 0.8% agrose gel to identify the recombined plasmid.Results: We constructed successfully a mammalian-expressing vector pEGFP-PDX-1.The recombined plasmid has the same open reading frame as pEGFP and perfect potential to translate and express in the mammalian cells.Conclusion: PDX1 was cloned into the C terminus of pEGFP-C1.Under the control of enhancer and promotor of pEGFP-C1,PDX-1 would be(expressed) as fusions with pEGFP-C1, increasing the translation efficiency of PDX-1 in mammalian cells without changing the structure and function of its translating protein.In addition,the recombined vector chooses GFP as a report gene to permit the positive cells to be selected in a simple way from the primary cell level.PDX-1;gene expression;pEGFP;PCR

     

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