冠心病相关基因肌细胞增强因子2A突变型真核表达载体的构建及核定位研究

The Construction of Eukaryotic Expression Vectors of Three MEF2A Mutants and Their Nuclear Localization in HEK293T Cells

目的构建冠心病相关基因肌细胞增强因子2A(MEF2A)的野生型及三种突变型真核表达载体,并观察其转染人胚肾293T(human embryo kidney293T,HEK293T)细胞后的核定位情况。方法通过对冠心病患者的MEF2A的第11外显子测序,选择在1258～1290(CAG)n和1291～1293CCG/-两个多态性位点存在△Q424～430、△Q430、△Q429△Q430△P431突变的患者,应用巢式PCR(Nested PCR)的方法扩增出MEF2A基因全长,构建野生型及三种突变型pEGFP-N1-MEF2A重组表达质粒,转染人胚肾上皮细胞系293T,用倒置荧光显微镜观察转染后MEF2A蛋白表达及核定位情况。结果经PCR和核酸序列分析证实各突变型MEF2A插入正确,成功构建pEGFP-N1-MEF2A重组表达质粒。经倒置荧光显微镜观察到转染野生型及突变型pEGFP-N1-MEF2A重组载体后,代表MEF2A蛋白的红色荧光均集中于细胞核内。结论 MEF2A野生型及△Q424～430、△Q430、△Q429△Q430△P431三种突变型载体均成功表达GFP融合MEF2A蛋白。三种突变型蛋白与野生型蛋白一样,均位于细胞核内,提示这三种突变对MEF2A的核定位无影响。

Objective To construct the eukaryotic vectors of three MEF2A(myocyte enhancer factor-2A)mutants and study their expressions in human embryo kidney 293T(HEK293T)cell line.Methods The mutations of the exon 11 of MEF2A gene in the patients with coronary artery disease were analyzed by polymerase chain reaction and DNA direct sequencing.The full length of wild type or mutated MEF2A genes were amplified by nested PCR,and then inserted into pEASY-T3 plasmid with T/A clone method;the constructs were verified by sequencing.The coding region of MEF2A(wild type or mutated)were subsequently sub-cloned into pEGFP-N1 fluorescent expression vector.The verified pEGFP-N1-MEF2A(wild type or mutated)was transfected into the HEK293T cell,the expression of which was observed using Fluorescence microscopy.Results The results from PCR and DNA sequencing analysis indicate the success in constructing corresponding expression vectors.Either wild type or mutant MEF2A is located in the nucleus of the transfected cells.Conclution Three mutants(△Q424～430,△Q430 and △Q429△Q430△P431)do not disrupt the distribution of MEF2A in HEK293T cells.