小鼠NF-κB p65 亚基真核表达质粒的构建.docVIP

  小鼠 NF-κB p65 亚基真核表达质粒的构建#  鲍斌，杨雪梅，刘健  ** （合肥工业大学 生物与食品工程学院） 5 10 15 20 25 30 35 摘要：作为一种代谢疾病，肥胖正在全球广泛蔓延，威胁人类健康。最新的研究表明，肥胖是一种复杂的 慢性低度炎性。巨噬细胞在脂肪沉积的过程中首先浸润到脂肪组织，直接参与到了相关炎性反应中。NF-κB 的激活与巨噬细胞所介导的炎性密切相关，p65 亚基是 NF-κB 信号调控下游靶基因转录的重要因子。构建 p65 的真核表达质粒可以为后续在脂肪组织巨噬细胞中研究 NF-κB 信号通路的调控奠定基础。因此，本研 究利用 PCR 技术扩增了 p65 基因，然后构建了 p65 的真核表达质粒（p65-EGFP），并通过 PCR 验证和酶切 验证了重组质粒的正确性。最后提取不含内毒素的质粒，转染 Hep G2 细胞，通过观测绿色荧光检测转染效 率。p65-EGFP 重组质粒的成功构建为后续开展巨噬细胞 NF-kB 信号通路的研究奠定了基础。 关键词：分子生物学；NF-kB; p65；重组质粒； 真核表达 中图分类号：Q786 Construction of mouse NF-κB p65 subunit expression plasmids BAO Bin, YANG Xuemei, LIU Jian (School of Biotechnology and Food Engineering, Hefei University of Technology) Abstract: As a kind of metabolic disease, obesity is globlly spreading, threatening human health. Recent researchs show that obesity is a kind of low chronic inflammatory reaction. As an important immune cells, macrophages firstly infiltrated into the adipose tissue in the process of fat deposition, and directly involved in the related inflammatory reaction. In macrophages, NF-κB activation is closely related to the rise of inflammatory, and p65 is quite important in the regulation of target genes transcription. Construting the p65 eukaryotic expression plasmid can promote the follow-up study of NF -kappa B signaling pathways in macrophages in adipose tissues. In this study, we amplified p65 with PCR, and then constructs the p65 of eukaryotic expression plasmid (p65 -pEGFP-C1). Then the recombinant plasmid is validated by enzyme digestion and PCR to verify the validity. Finally, we extract the recombinant plasmid, without endotoxin, and then transfect it to HepG2 cells. The transfection efficiency was detected by observing the green fluorescence. The success of reorganization of the recombinant plasmid (p65 -pEGFP-C1) can make great contribution to the subsequent research which is important to understand the mechanism of the NF-kB signaling pathway in macrophages in adipose tissues. Key words: Molecular Biol