利用Gateway技术构建神经元特异性人apoE4(1-272)真核表达质粒及鉴定

摘要

Objective To construct an expression plasmid of neuron-specific human apolipoprotein E4(1-272)fragment,and then identify its function by transfecting into N2a cells,which may provide a foundation for further study.Methods According to the Gateway technique,we designed the primer for apoE4(1-272)fragment,and get apoE4(1-272)fragment by PCR from the apoE4-EGFP plasmid.The apoE4(1-272)fragment was firstly cloned into pDONR221 vector by BP reaction,and then cloned into pRP.EX3d donor by LR reaction,meanwhile the neuron-specific promoter SYN1 was inserted prior to the apoE4(1-272)fragment,and the EGFP was also constructed by ligating the EGFP reporter group with the IRES element.The recombinant plasmid was transfected into N2a cells by LipofectamineTM2000,after 24 h,the transfection efficiency was observed by fluorescence microscopy and the expression level of apoE4(1-272)protein was detected by Western blotting.Results The expression plasmid of neuron-specific human apoE4(1-272)was successfully constructed,and the sequences of the core components were confirmed by sequencing.After being transfected into N2a cells,the newly constructed plasmid was successfully expressed,and the levels and molecular weight of apoE were consistent with the expected by Western blotting.Conclusion In this study,we have successfully constructed a recombinant expression plasmid of neuron-specific human apoE4(1-272)fragment,and identified its expression function in N2a cells,which would facilitate follow-up study for its function.%目的 构建神经元特异性人apoE4(1-272)片段的真核表达质粒,并转染小鼠神经母细胞瘤细胞(N2a细胞)验证其表达,为后续利用该质粒构建转基因小鼠,并探讨其作用奠定实验基础.方法 以同济医学院生物化学与分子生物学系实验室已有的人apoE4质粒为基础,利用Gateway技术设计引物,将人apoE4(1-272)片段克隆至pRP.EX3d载体上,并将神经元特异性启动子SYN1插入至人apoE4(1-272)片段之前,再借助IRES元件连接EGFP报告基因,构建pRP.EX3d-SYN1-apoE4(1-272)-IRES/EGFP重组表达质粒;经脂质体转染法将上述质粒转染至N2a细胞,转染24 h后,荧光显微镜观察转染效率,并采用Western blot检测apoE4(1-272)蛋白的表达水平.结果 成功构建神经元特异性表达人apoE4(1-272)的真核表达质粒,经测序鉴定各核心元件序列均正确无误,与预期结果相一致,且经转染N2a细胞观察到新构建质粒可顺利表达,Western blot检测结果显示apoE表达及分子量均与预期一致.结论 成功构建了神经元特异性人apoE4(1-272)片段的表达质粒,并初步鉴定了其表达功能,为后续开展相关研究奠定了实验基础.