人nestin第二内含子荧光素酶报告基因载体的构建及鉴定

目的构建含有人nestin第二内含子的荧光素酶报告基因并检测其活性。方法从NCBI数据库获得nestin的DNA全长序列（NC₀00001）,用vectorNTI软件分析找到其第二内含子位置,通过PCR的方法从人血全基因组DNA中钓取nestin的第二内含子全长片段,克隆到报告基因pGL3-promoter载体上,将报告基因载体转染至293T细胞,采用双荧光素酶报告基因系统评估第二内含子的活性。结果成功钓取nestin第二内含子片段,长度为1688bp,测定其DNA序列正确。瞬时转染293T细胞,经双荧光报告实验显示转染了pGL3-nes-promoter载体的细胞其相对荧光素酶活性相比于空载组,大约提高了3.39倍,差异有统计学意义（P<0.001）。结论成功构建了含有nestin第二内含子的荧光素酶报告载体,为进一步研究nestin的调控机制奠定基础。

The construction of the luciferase reporter vector with the second intron of nestin and identification of its activity

Objective To construct luciferase reporter vector with the second intron of nestin pGL3-nes-promoter and carry out its functional identification.Methods The whole DNA sequence of nestin was aquired from NCBI（NC₀00001）,and the position of its second intron was located.PCR was used to obtain the fragment of its second intron from genome DNA of the human blood cells.pGL3-promoter with the second intron of nestin was established and cotransfected into 293T cells.The dual lucifarese reportor assay was performed to detect the activity.Results The recombinant luciferase reporter vector was successfully constructed and was verified by PCR and sequencing.The dual lucifarese reportor assay showed a significant elevation of relative lucifarese activity after transfecting pGL3-nes-promoter（P<0.001）.Conclusion The luciferase reporter vector with the second intron of nestin is constructed successfully,which lay a experimental foundation for further research on the regulation of nestin.