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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: The coding sequence of Gadd45a was amplified from human embryonic stem cells by reverse transcription polymerase chain reaction (PCR) and cloned into pcDNA3.1 vector. The constructed expression plasmid pcDNA3.1-Gadd45a and the blank plasmid pcDNA3 .1 were transfected into Jurkat cells or normal human CD4 + T cells respectively, and the expression of Gadd45a mRNA and protein were detected by fluorescence quantitative RT-PCR and Western blot respectively. Results: Gadd45a expression plasmid pcDNA3.1-Gadd45a was successfully constructed. Both Jurkat and CD4 + T cells transfected with this plasmid showed Gadd45a overexpression. Conclusion: The construction of Gadd45a expression plasmid and its overexpression in human T cells provide the basis for further study on the role of Gadd45a in epigenetic mechanism.