人分化抑制因子Id-2、Id-3在E.coli中的高效表达及其多克隆抗体的制备

发布时间：2018-01-09 10:31

本文关键词：人分化抑制因子Id-2、Id-3在E.coli中的高效表达及其多克隆抗体的制备　出处：《黑龙江大学》2009年硕士论文　论文类型：学位论文

【摘要】：分化抑制因子又称DNA结合抑制因子,属于螺旋-环-螺旋蛋白,其对碱性螺旋-环-螺旋转录因子有负调控作用,可以抑制细胞分化、促进细胞增殖并且参与细胞周期调控过程。目前已知哺乳动物细胞含有4种亚型Id蛋白即Id-1~4。其中Id-2在控制细胞分化和增殖过程中发挥着重要的作用。它不但能对肝细胞转录因子进行负调节,抑制肝星状细胞分化,而且有研究表明Id-2在凋亡诱导的肝坏死中也扮演着重要角色。另外,Id-2表达随细胞衰老呈明显下降趋势,在破骨细胞、乳腺上皮、成纤维、角质、内皮等细胞中发现,其过度表达可以延迟细胞老化或导致细胞永生化。Id-2基因也具有癌基因的特性,其编码的Id-2蛋白能阻止细胞分化,促进细胞增殖,从而参与肿瘤的发生。而Id-3基因作为血清诱导的立即早期基因,在小鼠成纤维细胞系中被首次发现,其编码的Id-3蛋白能够参与多种细胞生物学过程,包括T细胞和B细胞的发育、骨骼肌的分化、血管平滑肌的增值、胚胎的神经形成、骨发生和肿瘤诱导的血管发生等。目前,Id蛋白已成为研究细胞生命过程及探寻治疗人类疾病有效靶向药物的一类重要分子。本研究在大肠杆菌中分别表达人Id-2与谷胱甘肽-S转移酶(GST)的融合蛋白及人Id-3与谷胱甘肽-S转移酶(GST)的融合蛋白,并在此基础上制备了抗人Id-2、Id-3的多克隆抗体。本试验从人乳腺癌组织中提取总RNA,应用RT-PCR方法扩增出Id-2、Id-3基因的编码序列,鉴定正确后将其克隆至表达载体pGEX-6P-1中,重组质粒经PCR、酶切、测序鉴定后,在大肠杆菌中经IPTG诱导表达获得GST-Id-2、GST-Id-3融合蛋白,SDS-PAGE分析表达产物。Western-Blot实验表明,通过亲和层析法纯化的GST-Id-2、GST-Id-3融合蛋白具有良好的免疫原性,然后以纯化的蛋白为抗原免疫健康新西兰白兔制备多克隆抗体,并利用酶联免疫吸附试验(ELISA)及琼脂免疫扩散试验检测抗体效价。本试验结果表明,经PCR、酶切、测序鉴定证明,Id-2、Id-3基因分别已正确克隆至pGEX-6P-1中,经IPTG诱导后,表达出相对分子质量为40 000的GST-Id-2融合蛋白及相对分子质量为39 000的GST-Id-3融合蛋白。Western-Blot、ELISA和琼脂双向扩散实验鉴定所制备的多克隆抗体可以与GST-Id-2和GST-Id-3发生特异性反应。本试验研究结果证明,Id-2、Id-3基因在大肠杆菌中获得了成功表达且成功制备了其相应的多克隆抗体,为检测Id-2、Id-3及其在各种组织中的表达提供了一种检测方法,也为分析Id-2、Id-3分子结构及抗原表位奠定了基础。
[Abstract]:The differentiation inhibitory factor called DNA binding inhibitory factor, which belongs to helix loop helix proteins, the basic helix loop helix transcription factor has a negative regulatory role, can inhibit cell differentiation and promote cell proliferation and cell cycle regulation. Known mammalian cells contain 4 subtypes of Id protein in Id-1~4. Id-2 the control of cell differentiation and proliferation plays an important role. It can not only negatively regulate transcription factor on liver cells, inhibiting hepatic stellate cell differentiation, and studies have shown that Id-2 induced apoptosis in hepatic necrosis also play an important role. In addition, the expression of Id-2 with cell senescence was significantly decreased in the osteoclasts, mammary epithelial cells, fibroblasts, keratinocytes, endothelial cells found, its overexpression can delay the aging of cells or cause characteristics of immortalized cell.Id-2 gene with cancer gene, its coding Code Id-2 protein can prevent cell differentiation, promote cell proliferation, and thereby involves in carcinogenesis. The Id-3 gene as immediate early genes induced by serum, a fibroblast cell line was first discovered in mice, its encoding Id-3 protein involved in many cellular processes, including T cell and B cell development, differentiation of bone muscle, vascular smooth muscle proliferation, embryonic neurogenesis, osteogenesis and tumor induced angiogenesis. At present, Id protein has become the research of cellular processes and explore the treatment of human diseases effectively targeted a kind of important molecular drugs.
This study were expressed in Escherichia coli Id-2 and glutathione -S transferase (GST) fusion protein and Id-3 and glutathione -S transferase (GST) fusion protein, and on the basis of the preparation of anti human Id-2 polyclonal antibody of Id-3. The total RNA was extracted from human breast cancer tissues the application of RT-PCR, amplified Id-2, encoding Id-3 gene sequence, after the identification of cloned into expression vector pGEX-6P-1. The recombinant plasmid was identified by PCR, enzyme digestion and sequencing, in Escherichia coli induced by IPTG expression of GST-Id-2, GST-Id-3 fusion protein, SDS-PAGE analysis showed that the expression product of.Western-Blot by affinity experiment. Chromatography purified GST-Id-2 GST-Id-3 fusion protein has good immunogenicity, and then purified protein antigen to immunize healthy New Zealand white rabbits to prepare polyclonal antibody, using enzyme-linked immunosorbent assay (ELISA) and Agar immunodiffusion test was used to detect antibody titer.
The test results show that by PCR, enzyme digestion and sequencing proved that Id-2 Id-3 gene was correctly cloned into pGEX-6P-1. After induced by IPTG, expression of the relative molecular mass of 40000 GST-Id-2 fusion protein and the relative molecular mass of 39000 GST-Id-3 fusion protein.Western-Blot, ELISA polyclonal antibody and double agar diffusion experiment identification of the prepared can react specifically with GST-Id-2 and GST-Id-3.
The test results show that the Id-2 Id-3 gene, obtained the corresponding polyclonal antibody was prepared successfully and successfully expressed in Escherichia coli, for the detection of Id-2, Id-3 and its expression in various tissues and provides a detection method for Id-2 analysis, Id-3 molecular structure and epitope laid the foundation.

【学位授予单位】：黑龙江大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R341

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