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

Construction of eukaryotic expression vector of rat Neurogenesin-1 gene and its expression in cos-7 cell

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 Neurogenesin-1 （ Ngl ） gene from the hippocampus of rat, then construct its eukaryotic expression vector pSecTag2/Hygro B-Ng1, and detect its expression in cos-7 cell so as to provide experimental evidence for gene therapy on spinal cord injury. Methods SD rat of postnatal for 7 days was selected and killed by dislocation of neck, the cerebrum was quickly taken out under cold condition, then dissected the hippocampus, after that, the total RNA of the hippocampus was extracted under the condition without RNA enzyme contamination. The complete encoded sequence of rat Ngl gene was amplified by reverse transcription-polymerase chain reaction（RT-PCR） from rat hippocampus. The amplified fragment was inserted into eukaryotic expression vector pSecTag2/Hygro B to construct the reeombined plasmid that encoded Ngl eDNA. Cos-7 cells were transfected mediated by liposome, then the expression protein was detected by Western blot. Results Ngl eDNA was successful obtained from rat hippocampus through RT-PCR. Then the recombinant plasmid was cut with restriction endonuclease Hind Ⅲ and Xho Ⅰ , then confirmation by sequencing and nucleotide homology analysed to GenBank, we successful obtained the recombinant plasmid pSecTag2/Hygro B-Ngl. Then the recombinant plasmid transfected cos-7 cells by liposome reagent. 48 hours after transfected, the recombined Ngl protein was detected, the specific 46 ku protein bands was detected. Conclusion We successful construct the eukaryotic expression vector of rat Ngl gene from rat hippocampus. After transfecting cos-7 cells, the recombinant Ng1 protein is detected.