大鼠Neurogenesin-1基因真核表达载体的构建及在cos-7细胞中的表达

发布时间：2018-01-04 12:09

本文关键词：大鼠Neurogenesin-1基因真核表达载体的构建及在cos-7细胞中的表达　出处：《安徽医科大学学报》2009年04期 　论文类型：期刊论文

【摘要】：目的克隆大鼠海马中Neurogenesin-1(Ng1)基因片段,构建pSecTag2/HygroB-Ng1真核表达载体,并检测其在cos-7细胞中的表达,为进一步研究该基因对脊髓神经干细胞分化的影响提供实验依据。方法在无RNA酶污染的条件下提取出大鼠海马总RNA。利用逆转录聚合酶链反应扩增出Ng1基因片段。将该基因片段连接到真核表达载体pSecTag2/HygroB,聚合酶链反应初步筛选,双酶切鉴定后送测序。将构建成功的重组真核表达载体转染入cos-7细胞,Westernblot鉴定重组Ng1蛋白的表达。结果逆转录聚合酶链反应成功获得大鼠Ng1cDNA。随机挑选10个重组真核表达载体的克隆,聚合酶链反应筛选出阳性克隆2个,经双酶切鉴定、测序及Blast分析鉴定重组质粒构建成功。脂质体介导转染cos-7细胞48h后,Westernblot鉴定重组Ng1蛋白在cos-7细胞中的表达,在46ku处出现阳性条带。结论大鼠海马Ng1基因的真核表达载体pSecTag2/HygroB-Ng1构建成功,转染cos-7细胞后能够表达重组Ng1蛋白。
[Abstract]:Objective to clone the neurogenesin-1 ng1) gene fragment from rat hippocampus and construct the eukaryotic expression vector of pSecTag2/HygroB-Ng1. Its expression in cos-7 cells was detected. Methods Total rat hippocampal RNA was extracted without RNA enzyme contamination and N was amplified by reverse transcriptase polymerase chain reaction (RT-PCR) in order to further study the effect of the gene on the differentiation of neural stem cells in spinal cord. The gene fragment was inserted into eukaryotic expression vector pSecTag2/HygroB. The recombinant eukaryotic expression vector was constructed and transfected into cos-7 cells. Results reverse transcriptase polymerase chain reaction (RT-PCR) was used to obtain rat Ng1cDNA. 10 recombinant eukaryotic expression vectors were randomly selected. Two positive clones were screened by polymerase chain reaction. The recombinant plasmid was identified by double enzyme digestion, sequencing and Blast analysis. The recombinant plasmid was transfected with liposome for 48 h. The expression of recombinant Ng1 protein in cos-7 cells was identified by Westernblot. Conclusion the eukaryotic expression vector pSecTag2/HygroB-Ng1 of rat hippocampal Ng1 gene was successfully constructed. The recombinant Ng1 protein was expressed after transfection of cos-7 cells.
【作者单位】：安徽医科大学第一附属医院骨科;安徽医科大学分子生物学教研室;
【基金】：国家自然科学基金资助项目(编号:30772201)
【分类号】：R651.2;R346
【正文快照】： 近年来,应用内源性神经干细胞治疗脊髓损伤越来越受到人们的关注。实验证明,成体动物脊髓实质中存在大量的神经干细胞,但这种细胞通常处于静止状态,只有在脊髓损伤后才活跃增生。然而,这种细胞大部分向星形胶质细胞的方向分化,参与局部的胶质瘢痕形成。如何通过有效的刺激使

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