重组Mash1慢病毒表达载体的构建及表达

发布时间：2018-01-18 12:32

  本文关键词：重组Mash1慢病毒表达载体的构建及表达　出处：《郑州大学》2006年硕士论文　论文类型：学位论文

  更多相关文章： Mash1 bHLH 基因转染 基因表达 RT-PCR

【摘要】：背景和目的： bHLH(basic helix-loop-helix，碱性螺旋-环-螺旋)转录调控因子是发育过程中转录网络的重要组成部分，，广泛参与神经和肌肉发生、细胞增殖分化、细胞谱系的决定和性别决定等基本生理过程。Mash1(mammalian achaete-scute homolog 1)基因属于bHLH转录调控因子家族成员之一，在神经发生、神经发育和神经分化过程中发挥重要的调控作用。Mash1基因功能缺失导致神经发育异常、神经元分化减少及特定亚型神经元缺失；而其过表达则可以促进P19细胞、神经干细胞和胚胎干细胞向神经细胞分化。由于Mash1基因的过表达研究多数应用的是质粒型表达载体，转染效率较低且不能稳定表达，如采用合适的载体将Mash1基因导入干细胞或体内或将转染Mash1基因的靶细胞植入损伤局部，使其在损伤部位持续分泌Mash1蛋白，必将有利于神经分化和神经损伤的修复。因此，为进一步探讨Mash1基因在神经发育和干细胞神经分化中的调控作用及为基因治疗神经系统疾病奠定基础，我们拟构建重组Mash1基因慢病毒表达载体。 实验方法： 1．Mash1cDNA克隆及序列分析 从小鼠胚胎中提取总RNA，逆转录为cDNA，PCR扩增获得小鼠Mash1基因，回收纯化；以T4连接酶连接Mash1基因片段和pGEM-T载体，转化感受态细菌JM109后经蓝白筛选和以T7／SP6为引物PCR扩增筛选鉴定重组克隆；小提重组克隆质粒DNA，应用限制性内切酶BamH Ⅰ和Xhol Ⅰ对其进一步酶切鉴定阳性克隆；对鉴定出的阳性重组质粒pGEM-T-Mash1进行基因测序。
[Abstract]:Background and purpose: BHLH(basic helix-loop-helix (basic helix-loop-helix) transcription regulator is an important part of transcription network during development. Extensive involvement in neurogenesis and musculogenesis, cell proliferation and differentiation. Basic physiological processes, such as cell lineage determination and sex determination. Mash1 mammalian achaete-scute homolog 1). Genes belong to the family of bHLH transcription regulators. Mash1 gene dysfunction in neurogenesis, neurogenesis and neural differentiation. The absence of Mash1 gene leads to abnormal neural development, reduced neuronal differentiation and specific subtype neuronal deletion. However, overexpression can promote the differentiation of P19 cells, neural stem cells and embryonic stem cells into neural cells. Because of the overexpression of Mash1 gene, most of them are plasmid expression vectors. Transfection efficiency is low and can not be stably expressed, such as the appropriate vector to transfer Mash1 gene into stem cells or in vivo or target cells transfected with Mash1 gene implanted into the local injury. The continuous secretion of Mash1 protein in the injured site will be conducive to nerve differentiation and nerve injury repair. To further explore the role of Mash1 gene in neural development and neural differentiation of stem cells and to lay a foundation for gene therapy of nervous system diseases. We intend to construct the recombinant Mash1 gene lentivirus expression vector. Experimental methods: 1. Mash1 cDNA was cloned and sequenced. Total RNAs were extracted from mouse embryos. Mouse Mash1 gene was amplified by reverse transcription-PCR with cDNA-DNA, and the mouse Mash1 gene was recovered and purified. Mash1 gene fragment and pGEM-T vector were ligated with T4 ligase. The recombinant clones were identified by blue and white screening and PCR amplification and screening using T7 / SP6 as primer. The recombinant plasmid DNA was isolated and identified by restriction endonuclease BamH 鈪

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