Gadd45a表达质粒的构建及在人类T细胞中的表达

摘要

目的:构建Gadd45a表达质粒,并诱导该质粒在人类T细胞中的表达.方法:采用反转录PCR法从人胚胎干细胞中扩增Gadd45a的蛋白质编码框,将之克隆至pcDNA3.1载体后,利用电穿孔方法将构建好的表达质粒pcDNA3.1-Gadd45a和空白质粒pcDNA3.1分别转染至Jurkat细胞或正常人CD4+T细胞,分别用荧光定量RT-PCR和Western blot的方法检测转染后细胞Gadd45a mRNA和蛋白的表达.结果:成功构建Gadd45a表达质粒pcDNA3.1-Gadd45a,转染该质粒的Jurkat和正常人CD4+T细胞均显示Gadd45a过表达.结论:Gadd45a表达质粒的构建及诱导其在人T细胞中的过表达为进一步研究Gadd45a在表观遗传学机制中的作用提供了研究基础.%Objective To construct Gadd45a expression plasmid and induce its expression in human T cells.Methods Gadd45a was amplified by reverse transcription PCR from human embryonic stem cells, and cloned into the pcDNA 3.1 vector.The recombinant plasmid or blank plasmid was transfected into Jurkat cells or normal human CD4 +T cells using electroporation, and the expression of Gadd45a was detected by quantitative RT-PCR and Western blot.Results Human Gadd45a expression plasmid was constructed successfully.Gadd45a was overexpressed both in Jurkat cells and normal human CD4 + T cells after these cells were transfected with pcDNA 3.1-Gadd45a.Conclusion The construction of Gadd45a expression plasmid and induction of Gadd45a overexpression in human T cells lay the foundation for further research on the role of Gadd45 a in the epigenetic mechanism.