人巨噬细胞移动抑制因子的原核表达及其单克隆抗体的制备
发布时间：2018-11-21 10:58
【摘要】：人巨噬细胞移动抑制因子(Human macrophage migration inhibitory factor, hMIF)是一种重要的免疫调控细胞因子,组成性表达于全身多种组织,其主要来源是垂体前叶细胞、活化的T淋巴细胞、单核巨噬细胞等。hMIF基因编码区含353bp,编码由115个氨基酸残基组成、分子量约为12.5kD的非糖基化蛋白。除细胞因子的基本功能外,hMIF还具有类似激素、生长因子、应激反应蛋白及糖基化抑制因子(glycosylated inhibitory factors, GIF)等的多重作用,并可调节机体的免疫应答。近年来研究发现和证实,hMIF可促进感染性休克、风湿性关节炎等感染性、自体免疫性疾病以及肿瘤的发生发展。此外,hMIF还可加速创伤后的组织修复。故本文hMIF的原核表达和抗hMIF单克隆抗体的制备研究将为上述疾病的诊断和治疗提供新的机遇。 主要的研究内容和结果: 1.hMIF基因的克隆和原核表达 (1)根据GenBank发表的hMIF cDNA序列设计了一对特异引物,经RT-PCR从人乳腺癌细胞株MDA-MB453和人乳腺癌组织扩增hMIF cDNA,琼脂糖凝胶电泳发现扩增出全长为353bp的hMIF基因片段;凝胶回收试剂盒回收PCR产物并将其构建到原核表达载体pET11b中,测序表明所克隆到的hMIF序列与GenBank发表的hMIF cDNA序列完全一致。 (2)重组质粒pET11b/hMIF转化大肠杆菌BL21(DE3),按IPTG的作用时间分组诱导hMIF蛋白的表达;Tricine SDS-PAGE可见,与未诱导组相比,IPTG诱导组细菌有一条分子量约为12.5kD的特异蛋白条带,与hMIF蛋白的预计分子量相符;诱导6h时蛋白表达量最高,约占细菌总蛋白的30%。将表达菌破菌离心后分别取细菌上清和沉淀电泳,证实该蛋白为可溶性表达。 (3)将BL21重组菌(pET11b/hMIF)诱导发酵,超声破菌;1g湿菌经阳离子交换和疏水层析可得到约53mg纯度为95.40%的hMIF,得率约为17.7%,纯化的蛋白浓度为1.145g/L,Western blotting结果表明纯化的hMIF蛋白能与鼠抗hMIF单克隆抗体形成特异性条带。
[Abstract]:Human macrophage migration inhibitory factor (Human macrophage migration inhibitory factor, hMIF) is an important immunomodulatory cytokine expressed in various tissues of the whole body. It is mainly derived from anterior pituitary cells and activated T lymphocytes. The coding region of hMIF gene contains 353bp. it is composed of 115 amino acid residues and its molecular weight is about the non-glycosylated protein of 12.5kD. In addition to the basic functions of cytokines, hMIF also has multiple effects such as hormone, growth factor, stress response protein and glycosylation inhibitor (glycosylated inhibitory factors, GIF), and can regulate the immune response of the body. In recent years, it has been found and confirmed that hMIF can promote the development of septic shock, rheumatoid arthritis, autoimmune disease and tumor. In addition, hMIF can accelerate tissue repair after trauma. Therefore, the prokaryotic expression of hMIF and the preparation of monoclonal antibody against hMIF will provide a new opportunity for the diagnosis and treatment of these diseases. Main contents and results: cloning and prokaryotic expression of 1.hMIF gene (1) A pair of specific primers were designed according to the hMIF cDNA sequence published by GenBank. HMIF cDNA, agarose gel electrophoresis was used to amplify the 353bp gene fragment from human breast cancer cell line MDA-MB453 and human breast cancer tissue by RT-PCR. The gel recovery kit recovered the PCR product and constructed it into the prokaryotic expression vector pET11b. The sequence of the cloned hMIF was completely consistent with the hMIF cDNA sequence published by GenBank. (2) the recombinant plasmid pET11b/hMIF was transformed into Escherichia coli BL21 (DE3), and the expression of hMIF protein was induced according to the time of IPTG. Tricine SDS-PAGE showed that the IPTG induced bacteria had a specific protein band with a molecular weight of about 12.5kD, which was consistent with the predicted molecular weight of the hMIF protein, and the protein expression was the highest at 6 h after induction, accounting for about 30% of the total bacterial protein. Bacterial supernatant and precipitation electrophoresis were obtained after centrifugation of the expressed bacteria and the protein was confirmed as soluble expression. (3) the BL21 recombinant bacteria (pET11b/hMIF) were induced to ferment, and the ultrasound was used to break the bacteria. By cation exchange and hydrophobic chromatography, 1g wet bacteria could get about 95.40% 53mg, and the yield of hMIF, was about 17.7g / L, and the concentration of purified protein was 1.145g / L. Western blotting results showed that the purified hMIF protein could form a specific band with mouse anti-hMIF monoclonal antibody.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392.1
本文编号：2346770
[Abstract]:Human macrophage migration inhibitory factor (Human macrophage migration inhibitory factor, hMIF) is an important immunomodulatory cytokine expressed in various tissues of the whole body. It is mainly derived from anterior pituitary cells and activated T lymphocytes. The coding region of hMIF gene contains 353bp. it is composed of 115 amino acid residues and its molecular weight is about the non-glycosylated protein of 12.5kD. In addition to the basic functions of cytokines, hMIF also has multiple effects such as hormone, growth factor, stress response protein and glycosylation inhibitor (glycosylated inhibitory factors, GIF), and can regulate the immune response of the body. In recent years, it has been found and confirmed that hMIF can promote the development of septic shock, rheumatoid arthritis, autoimmune disease and tumor. In addition, hMIF can accelerate tissue repair after trauma. Therefore, the prokaryotic expression of hMIF and the preparation of monoclonal antibody against hMIF will provide a new opportunity for the diagnosis and treatment of these diseases. Main contents and results: cloning and prokaryotic expression of 1.hMIF gene (1) A pair of specific primers were designed according to the hMIF cDNA sequence published by GenBank. HMIF cDNA, agarose gel electrophoresis was used to amplify the 353bp gene fragment from human breast cancer cell line MDA-MB453 and human breast cancer tissue by RT-PCR. The gel recovery kit recovered the PCR product and constructed it into the prokaryotic expression vector pET11b. The sequence of the cloned hMIF was completely consistent with the hMIF cDNA sequence published by GenBank. (2) the recombinant plasmid pET11b/hMIF was transformed into Escherichia coli BL21 (DE3), and the expression of hMIF protein was induced according to the time of IPTG. Tricine SDS-PAGE showed that the IPTG induced bacteria had a specific protein band with a molecular weight of about 12.5kD, which was consistent with the predicted molecular weight of the hMIF protein, and the protein expression was the highest at 6 h after induction, accounting for about 30% of the total bacterial protein. Bacterial supernatant and precipitation electrophoresis were obtained after centrifugation of the expressed bacteria and the protein was confirmed as soluble expression. (3) the BL21 recombinant bacteria (pET11b/hMIF) were induced to ferment, and the ultrasound was used to break the bacteria. By cation exchange and hydrophobic chromatography, 1g wet bacteria could get about 95.40% 53mg, and the yield of hMIF, was about 17.7g / L, and the concentration of purified protein was 1.145g / L. Western blotting results showed that the purified hMIF protein could form a specific band with mouse anti-hMIF monoclonal antibody.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392.1
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