携带增强绿色荧光蛋白基因的Id2真核表达载体构建

Construction of Id2 eukaryotic expression vector containing green fluorescence protein gene

目的:构建大鼠Id2基因真核荧光表达载体,为骨骼肌的组织工程研究提供有效的分子工具。方法:利用RT-PCR的方法扩增出Id2全长cDNA,利用T4DNA连接酶将载体pGEM-T和Id2cDNA进行连接,构建克隆载体,经限制性内切酶EcoRI酶切pGEM-Id2克隆载体和pEGFP-C2真核表达载体,构建出重组真核表达载体pEGFP-C2-Id2,经酶切分析、PCR鉴定及DNA测序证实cDNA片段大小和序列的正确性;并通过细胞转染技术将Id2基因导入L6成肌细胞中。结果:经酶切分析和序列测定证实pEGFP-C2-Id2含大小正确的正向Id2cDNA片段,获取了转染外源性Id2基因的L6细胞。结论:我们成功构建了同时携带有G418筛选位点和增强绿色荧光蛋白的Id2真核表达载体。

Objective: To construct the eukaryotic expression vector of rat Id2 for further study on skeletal muscle tissue engineering. Methods: RT-PCR method was used to amplify the entire Id2 cDNA. The pGEM-T and Id2 cDNA were ligated by T4 DNA ligase. The cloning vectors and the pEGFP-C2 (eukaryotic expression vector) were first cut by EcoR I, and then ligated with Id2 by T4 DNA ligase again. The enzyme analysis and DNA sequencing was used to confirm the recombined vector. The pEGFP-C2-Id2 vector was transferred into L6 cells. Results: The results of enzyme analysis and DNA sequencing analysis confirmed that the right Id2 gene was cloned. The Id2 transferred L6 cells were gained. Conclusions: pEGFP-C2-Id2 eukaryotic expressing vector, which contains G418 selected site and green fluorescence protein gene, is successfully constructed.