IL-37融合EGFP真核表达载体的构建及其在293T细胞中的表达

发布时间：2018-03-11 20:43

本文选题：白介素-　切入点：绿色荧光蛋白　出处：《实用医学杂志》2015年09期 　论文类型：期刊论文

【摘要】：目的:构建白介素-37(interleukin-37,IL-37)与增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)融合基因真核表达载体,并检测其在293T细胞中的表达。方法 :通过PCR分别扩增出IL-37和EGFP基因,经酶切和测序鉴定正确后,克隆入真核表达载体pc DNA3.1,用Linker(G4S)3连接2个目的基因片段,构建IL-37-GFP融合基因真核表达质粒pc DNA3.1/IL-37-EGFP,将其转染到293T细胞中,观察荧光产生情况,用实时荧光定量RT-PCR和Western blot检测IL-37的表达情况。结果:经酶切和测序鉴定,由linker连接的IL-37-GFP融合基因正确克隆入表达载体pc DNA3.1,重组表达质粒pc DNA3.1/IL-37-EGFP转染293T细胞后,用荧光显微镜检测显示荧光蛋白在细胞中表达,RT-PCR与Western blot均检测到高水平IL-37 m RNA和IL-37蛋白的表达。结论:成功构建了IL-37-融合报告基因EGFP的真核表达载体,并在293T细胞中得到了有效表达,为进一步研究IL-37的功能和表达调控奠定了实验基础。
[Abstract]:Objective: to construct the eukaryotic expression vector of interleukin-37 interleukin-37 (IL-37) fusion gene with enhanced green fluorescent protein (EGFP), and to detect its expression in 293T cells. Methods: IL-37 and EGFP genes were amplified by PCR and identified correctly by restriction endonuclease digestion and sequencing. The eukaryotic expression vector pcDNA3.1 was cloned, and two target gene fragments were ligated with Linker(G4S)3. The eukaryotic expression plasmid of IL-37-GFP fusion gene, pcDNA3.1% IL-37-EGFP, was constructed and transfected into 293T cells, and the fluorescence production was observed. The expression of IL-37 was detected by real-time fluorescence quantitative RT-PCR and Western blot. Results: by restriction endonuclease digestion and sequencing, the IL-37-GFP fusion gene linked with linker was correctly cloned into the expression vector PC DNA3.1, and the recombinant expression plasmid PC DNA3.1/IL-37-EGFP was transfected into 293T cells. Fluorescence microscopy showed that high level IL-37 m RNA and IL-37 protein were detected by RT-PCR and Western blot. Conclusion: the eukaryotic expression vector of IL-37- fusion reporter gene EGFP was constructed successfully. It was expressed effectively in 293T cells, which laid an experimental foundation for further study on the function and regulation of IL-37.
【作者单位】：广东省医学分子诊断重点实验室广东医学院医学检验学院;
【基金】：国家自然科学基金资助项目(编号:81302244) 广东省大学生创新训练计划项目(编号:1057113038)
【分类号】：R363

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