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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。
Objective To clone the gene fragment of Neurogenesin-1 (Ng1) in rat hippocampus and construct the eukaryotic expression vector pSecTag2 / HygroB-Ng1 and to detect its expression in cos-7 cells. To further investigate the effect of this gene Provide experimental basis. Methods Total RNA was extracted from rat hippocampus without RNase contamination. The Ng1 gene fragment was amplified by reverse transcription polymerase chain reaction. The gene fragment was ligated into the eukaryotic expression vector pSecTag2 / HygroB, and was initially screened by polymerase chain reaction (PCR) and double enzyme digestion. The constructed recombinant eukaryotic expression vector was transfected into cos-7 cells and the expression of recombinant Ng1 protein was identified by Western blot. Results The rat Ng1 cDNA was successfully obtained by reverse transcription polymerase chain reaction. The clones of 10 eukaryotic expression vectors were randomly selected. Two positive clones were screened by polymerase chain reaction (PCR). The two clones were identified by restriction enzyme digestion, DNA sequencing and Blast analysis. The recombinant plasmids were successfully constructed. The expression of recombinant Ng1 protein in cos-7 cells was identified by Western blot after transfection of COS-7 cells with liposome for 48h, and a positive band appeared at 46ku. Conclusion The eukaryotic expression vector pSecTag2 / HygroB-Ng1 of hippocampal Ng1 gene was successfully constructed and transfected into cos-7 cells, which can express recombinant Ng1 protein.