| 常新荣, 陈玉英, 常平安.小鼠烟酰胺N-甲基转移酶基因的克隆及其表达.[J].中南医学科学杂志.,2012,40(5):457-460. |
| 小鼠烟酰胺N-甲基转移酶基因的克隆及其表达 |
| Cloning and Expression of Mouse Nicotinamide N-methyltransferase |
| 投稿时间：2012-04-16 |
| DOI： |
| 中文关键词: 烟酰胺N-甲基转移酶 表达质粒 编码cDNA 细胞定位 |
| 英文关键词:nicotinamide N-methyltransferase expressing plasmid coding cDNA cellular location |
| 基金项目:重庆市自然科学基金项目（CSTC 2010BB5410，2011JJA10110）；重庆市高校优秀人才计划支持项目. |
|
| 摘要点击次数: 884 |
| 全文下载次数: 972 |
| 中文摘要: |
| 目的克隆和表达小鼠烟酰胺N-甲基转移酶（NNMT）基因。方法设计特异引物，利用逆转录PCR克隆小鼠NNMT编码cDNA序列，并进行序列测定；然后通过PCR扩增，双酶切回收cDNA片段定向重组到载体pEGFP-N3中，采用PCR和酶切鉴定重组表达质粒；最后将其转染到真核细胞中，通过Western杂交检测其表达，用激光共聚焦显微镜检测其细胞分布。结果经RT-PCR获得了小鼠NNMT的编码cDNA序列，经测序证实克隆序列完全正确；PCR和酶切鉴定表明绿色荧光蛋白(GFP)标记的NNMT表达质粒pNNMT-GFP成功构建；转染至HEK293T细胞中，Western杂交检测到绿色荧光蛋白融合的NNMT特异表达，显微观察表明NNMT分布在细胞质中。结论成功构建了NNMT的表达质粒，并在小鼠中实现其表达。 |
| 英文摘要: |
| ObjectiveAim To clone and express mouse nicotinamide N-methyltransferase (NNMT).MethodsThe coding cDNA sequence of mouse NNMT gene was firstly cloned by reverse transcription-polymerase chain reaction (RT-PCR) using gene specific primers and certified by DNA sequencing. NNMT coding cDNA was amplified by PCR using primers containing specific restrict endonuclease site, and then inserted into the expression vector pEGFP-N3 to construct a recombinant plasmid, which was confirmed by PCR and restrict endonuclease； lastly the construction was transfected into eukaryotic cells and its expression and subcellular location were detected by western blotting and confocal microscopy respectively. ResultsThe coding cDNA sequence of mouse NNMT was obtained by RT-PCR and had no mutation as certified by DNA sequencing. The recombinant plasmid, pNNMT-GFP, to express NNMT tagged with GFP was generated, which was confirmed by PCR and cut with restrict endonuclease. pNNMT-GFP was transfected into HEK293T cells and NNMT fused with GFP was specifically detected by western blotting. The subcellular distribution of NNMT was in cytoplasm. ConclusionThe recombinant plasmid, pNNMT-GFP, to express NNMT tagged with GFP was successfully constructed and expressed in mammalian cells. |
| 查看全文 查看/发表评论 下载PDF阅读器 |
| 关闭 |