人巨噬細(xì)胞移動(dòng)抑制因子的原核表達(dá)及其單克隆抗體的制備
發(fā)布時(shí)間：2018-11-21 10:58
【摘要】：人巨噬細(xì)胞移動(dòng)抑制因子(Human macrophage migration inhibitory factor, hMIF)是一種重要的免疫調(diào)控細(xì)胞因子,組成性表達(dá)于全身多種組織,其主要來源是垂體前葉細(xì)胞、活化的T淋巴細(xì)胞、單核巨噬細(xì)胞等。hMIF基因編碼區(qū)含353bp,編碼由115個(gè)氨基酸殘基組成、分子量約為12.5kD的非糖基化蛋白。除細(xì)胞因子的基本功能外,hMIF還具有類似激素、生長(zhǎng)因子、應(yīng)激反應(yīng)蛋白及糖基化抑制因子(glycosylated inhibitory factors, GIF)等的多重作用,并可調(diào)節(jié)機(jī)體的免疫應(yīng)答。近年來研究發(fā)現(xiàn)和證實(shí),hMIF可促進(jìn)感染性休克、風(fēng)濕性關(guān)節(jié)炎等感染性、自體免疫性疾病以及腫瘤的發(fā)生發(fā)展。此外,hMIF還可加速創(chuàng)傷后的組織修復(fù)。故本文hMIF的原核表達(dá)和抗hMIF單克隆抗體的制備研究將為上述疾病的診斷和治療提供新的機(jī)遇。 主要的研究?jī)?nèi)容和結(jié)果: 1.hMIF基因的克隆和原核表達(dá) (1)根據(jù)GenBank發(fā)表的hMIF cDNA序列設(shè)計(jì)了一對(duì)特異引物,經(jīng)RT-PCR從人乳腺癌細(xì)胞株MDA-MB453和人乳腺癌組織擴(kuò)增hMIF cDNA,瓊脂糖凝膠電泳發(fā)現(xiàn)擴(kuò)增出全長(zhǎng)為353bp的hMIF基因片段;凝膠回收試劑盒回收PCR產(chǎn)物并將其構(gòu)建到原核表達(dá)載體pET11b中,測(cè)序表明所克隆到的hMIF序列與GenBank發(fā)表的hMIF cDNA序列完全一致。 (2)重組質(zhì)粒pET11b/hMIF轉(zhuǎn)化大腸桿菌BL21(DE3),按IPTG的作用時(shí)間分組誘導(dǎo)hMIF蛋白的表達(dá);Tricine SDS-PAGE可見,與未誘導(dǎo)組相比,IPTG誘導(dǎo)組細(xì)菌有一條分子量約為12.5kD的特異蛋白條帶,與hMIF蛋白的預(yù)計(jì)分子量相符;誘導(dǎo)6h時(shí)蛋白表達(dá)量最高,約占細(xì)菌總蛋白的30%。將表達(dá)菌破菌離心后分別取細(xì)菌上清和沉淀電泳,證實(shí)該蛋白為可溶性表達(dá)。 (3)將BL21重組菌(pET11b/hMIF)誘導(dǎo)發(fā)酵,超聲破菌;1g濕菌經(jīng)陽離子交換和疏水層析可得到約53mg純度為95.40%的hMIF,得率約為17.7%,純化的蛋白濃度為1.145g/L,Western blotting結(jié)果表明純化的hMIF蛋白能與鼠抗hMIF單克隆抗體形成特異性條帶。
[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.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2005
【分類號(hào)】：R392.1
本文編號(hào)：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.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2005
【分類號(hào)】：R392.1
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