4ARE强化的双荧光蛋白报告基因载体的构建及鉴定

目的:构建4ARE强化的红、绿双荧光蛋白报告基因载体。方法:人工合成3对ARE序列，经退火和磷酸化后插入pARE-TK-GFP载体，构建成p4ARE-TK-GFP载体。以质粒pDsRed2-N1为模板，PCR扩增红色荧光蛋白及其启动子序列与pMD18-T载体连接，构建成pMD-DsRed载体。用Ade I酶和BspT I酶对载体pMD-DsRed和p4ARE-TK-GFP进行双酶切，连接产物转化大肠埃希菌DH5α 感受态细胞后，挑取阳性克隆子进行质粒抽提酶切及测序鉴定。结果:经酶切及DNA测序证实，目的片段ARE及DsRed的序列完全正确，重组双荧光蛋白报告基因载体成功转入DH5α。结论:成功构建4ARE强化的双荧光蛋白报告基因载体并在DH5α内表达，为进一步研究ARE的调控作用奠定了基础。

Construction and identification of dual fluorescent protein reporter gene vector enhanced by four copies ARE

Objective:To construct the dual fluorescent protein reporter gene vector enhanced by four copies ARE.Methods:Three synthetic oligonucleotide ARE motifs were annealed and purified then inserted into the vector pARE-TK-GFP to construct the recombinant reporter vector p4ARE-TK-GFP.A red fluorescent protein and promoter fragment was cloned into the vector pMD18-T from pDsRed2-N1 to construct the vector pMD-DsRed.The vectors pMD-DsRed and p4ARE-TK-GFP were digested by Ade I and BspT I.The red fluorescent protein and promoter fragments were cloned into the vector p4ARE-TK-GFP to construct the dual fluorescent protein reporter gene vector.Results:The recombinant dual fluorescent protein reporter gene vector p4ARE-TK-GFP/DsRed was identified that it was successfully transformed into E.coli DH5α and expressed by restriction enzyme digestion and DNA sequencing.Conclusion:The dual fluorescent protein reporter gene vector enhanced by four copies ARE was successfully constructed and expressed in E.coli DH5α.This study lays a foundation for further research in the function of ARE.