从噬菌体十二肽库中筛选NF-κB p65亚基亲和肽的研究

发布时间：2018-04-17 19:45

本文选题：核因子-κB(NF-κB) + p65亚基　；参考：《中国人民解放军军事医学科学院》2005年硕士论文

【摘要】：核因子-κB(nuclear factor-κB,NF-κB)是一种重要的核转录因子,可被多种刺激因素激活,并易位至细胞核,与相应的靶基因结合,启动基因转录。NF-κB的靶基因包括编码细胞因子、粘附分子、诱导型一氧化氮合酶等的基因,因此与多种炎性疾病、自身免疫性疾病和肿瘤的发生有关。NF-κB已成为了疾病治疗的重要靶标。本实验室在化学性急性肺损伤研究研究中也发现,全氟异丁烯吸入中毒导致的急性肺损伤,其中NF-κB也表现为过度表达,表明NF-κB的异常激活与急性肺损伤的发生密切相关。噬菌体展示技术是寻找小肽拮抗分子的最有效的方法之一,该技术已在蛋白质结构研究、抗原表位分析、人工抗体制备、细胞因子拮抗剂的研究和寻找细胞内传递蛋白等研究中有广泛的应用,并在寻找导向小分子方面已取得了令人振奋的进展。因此本研究通过基因工程技术构建重组表达的NF-κB p65的表达载体,并对表达产物进行分离、纯化。以表达的GST-NF-κB p65(GST-p65)融合蛋白为靶标,利用噬菌体十二随机肽库筛选NF-κB p65亚基结合肽,为寻找NF-κB特异性抑制剂奠定基础。根据人NF-κB p65亚基基因序列设计引物,以pEGFP/p65质粒为模板PCR扩增得到NF-κB p65基因,将该基因克隆到克隆载体pGEM-T,获得克隆重组质粒pGEM-T/p65。将克隆重组质粒中的目的基因片段进行序列测定并与人NF-κB p65基因进行比较,结果序列一致读码框没有发生改变。从克隆重组质粒上切下目的基因片段,将该基因克隆到谷胱甘肽S转移酶(glutathione S-transferase,GST)融合表达载体pGEX-4T-2上,在大肠杆菌BL21中经IPTG诱导表达。得到的GST-p65
[Abstract]:Nuclear factor-魏 B(nuclear factor- 魏 B (NF- 魏 B) is an important nuclear transcription factor, which can be activated by many stimuli and translocated to the nucleus, which binds to the corresponding target gene. The target gene that activates the gene transcription. NF- 魏 B includes encoded cytokines and adhesion molecules.Inducible nitric oxide synthase (iNOS) and other genes are involved in many inflammatory diseases, autoimmune diseases and tumorigenesis. NF- 魏 B has become an important target of disease treatment.In our laboratory, we also found that the acute lung injury induced by perfluoroisobutene inhalation was also overexpression of NF- 魏 B, which indicated that the abnormal activation of NF- 魏 B was closely related to the occurrence of acute lung injury.Phage display is one of the most effective methods to search for small peptide antagonist molecules. It has been used in protein structure analysis, antigen epitope analysis and preparation of artificial antibodies.The study of cytokine antagonists and the search for intracellular transfer proteins have been widely used, and have made exciting progress in the search for targeted small molecules.Therefore, the recombinant expression vector of NF- 魏 B p65 was constructed by genetic engineering, and the expression product was isolated and purified.The fusion protein of GST-NF- 魏 B p65 (GST-p65) was used to screen NF- 魏 B p65 subunit binding peptide using phage 12 random peptide library, which laid the foundation for searching for NF- 魏 B specific inhibitor.According to the sequence of human NF- 魏 B p65 subunit gene, the NF- 魏 B p65 gene was amplified by PCR using pEGFP/p65 plasmid as template. The gene was cloned into the clone vector pGEM-T, and the cloned recombinant plasmid pGEM-T / p65 was obtained.The target gene fragment in the cloned recombinant plasmid was sequenced and compared with the human NF- 魏 B p65 gene.The target gene fragment was cut off from the cloned recombinant plasmid and cloned into the fusion expression vector pGEX-4T-2 of glutathione S-transferase (GST), which was induced by IPTG in Escherichia coli BL21.Obtained GST-p65
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：Q789

【共引文献】