This work aims to clone NeuroD2 gene,construct the eukaryotic expression vector pIRES2-AcGFP1-ND2,and analyze its expression in mouse MSCs. The rat NeuroD2 gene was cloned by RT-PCR,and the protein structure,functional domain,cell localization and homology with other species were analyzed. The constructed pIRES2-AcGFP1-ND2 expression vector was transfected to mouse bone marrow mesenchymal stem cells(MSCs). The expression of the recombinant plasmid in the mouse MSCs was detected by real time-PCR,Western blot and immunofluorescence. The results showed that,the length of CDS in rat NeuroD2 gene was 1149 bp,and it encoded 382 amino acids, belonging to bHLH family. The secondary structure mainly composed of the α-helices and random coil. Tertiary structure of NeuroD2 was a"helix-loop-helix"structure and mainly localized in nucleus. The NeuroD2 protein of the experimental rat(Rattus norvegicus)shared 99%, 98% and 97.7% homology with Mus musculus,Macaca mulatta and Homo sapiens,respectively. The results of real-time PCR,Western blot and immunofluorescence indicated that the expression levels of NueorD2 mRNA and protein in transfected group were significantly higher than that in control group(P < 0.05). In conclusion,the eukaryotic expression vector pIRES2-AcGFP1-ND2 was constructed successfully.%克隆大鼠NeuroD2基因,旨在构建pIRES2-AcGFP1-ND2真核表达载体并检测其在小鼠MSCs中的表达.采用RT-PCR技术克隆大鼠NeuroD2基因,分析该蛋白质结构、功能域、细胞定位及与其他物种同源性;构建pIRES2-AcGFP1-ND2表达载体并转染小鼠骨髓间充质干细胞(MSCs);采用Real-time PCR、免疫荧光及Western blot技术检测重组质粒在小鼠MSCs中的表达.结果表明,大鼠NeuroD2基因CDS区全长1149 bp,共编码382个氨基酸,属于bHLH家族,二级结构以 α-螺旋和无规卷曲为主,空间结构呈现近似"螺旋-环-螺旋(HLH)"结构,主要分布在细胞核,氨基酸序列与家鼠(99%)、罗猴(98%)、人(97.7%)同源性较高;Real-time PCR结果表明转染组NeuroD2基因表达量显著高于对照组(P<0.05);免疫荧光及Western blot结果显示转染组NeuroD2蛋白表达量显著高于对照组(P<0.05).成功克隆大鼠NeuroD2基因并构建了pIRES2-AcGFP1-ND2真核表达载体.
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