小鼠烟酰胺单核苷酸腺苷酰转移酶1基因的克隆与表达检测

The cloning of mouse nicotinamide mononucleotide adenylyl-transferase gene and the detecting of its expression

目的检测真核表达克隆pcDNA3．1烟酰胺单核苷酸腺苷酰转移酶1（NMNAT1）的水平和有效性。方法采用PCR的方法钓取小鼠源NMNAT1基因的cds全长片段，构建到T载体中，并转接到pcDNA3．1中；同时设计引物进行全基因测序；采用常规的脂质体转染方法转染入Hela细胞系中表达小鼠源NMNAT1基因，通过qPCR和Western blot的方法检测克隆pcDNA3．1-NMNAT1的表达水平和表达有效性，并同时建立小鼠源NMNAT1基因的qPCR和Western blot检测方法。结果成功钓取了小鼠源NMNAT1基因，测序结果显示与数据库序列完全匹配，pcDNA3．1-NMNAT1通过脂质体转染入Hela细胞系，48h以后收取细胞，经过反转录以后进行qPCR检测和Western blot检测，结果显示表达克隆pCDNA3．1-NMNAT1能同时在tuRNA水平和蛋白水平高表达小鼠NMNAT1基因。结论成功从小鼠脑cDNA文库中克隆了NMNAT1基因的全长cDNA，与Pubmed序列完全一致，并构建到真核表达载体上正确表达NMNAT1蛋白质。

Objective To construct eukaryotic expressing vector of the mouse NMNAT1 （nicotinamide mononucleotide adenylyl-transferase） gene and examine its ability to express the NMNAT1 gene in Hela cells. Methods The full-length NMNAT1 eDNA sequence was amplified by PCR and cloned into the plasmid of T-vector and then to pcDNA3.1 construct. The recombinant plasmid pcDNA3.1-NMNAT1 was identified by DNA sequencing and then transfected with Lipofectamine2000 into Hela cells. The expression of NMNAT1 was detected by real time quantitative PCR （qPCR） and Western blot after 48 h transfection. Results The recombinant eukaryotic vector carrying NMNAT1 gene was constructed successfully in a match with database and this vector could up-regulate the expression of the NMNAT1 gene both in mRNA and protein levels in Hela cells. Conclusions The eukaryotic vector carrying NMNAT1 gene （pcDNA3.1-NMNAT1） enhances the expression of NMNAT1 gene.