小鼠IL-33的原核表达、纯化及其多克隆抗体的制备与应用研究

发布时间：2018-08-04 10:26

【摘要】：白细胞介素(IL)-33是2005年Schmitz等使用计算机序列分析、发现并鉴定的一种前炎症细胞因子。它的基因序列和结构与IL-1家族成员IL-1β和IL-18最为相似,属于IL-1家族的一个新成员,也被称为IL-1F11。IL-33 mRNA在小鼠胃、肺、脊髓、脑、皮肤等组织及静止树突状细胞、活化的巨噬细胞高表达,在人平滑肌细胞及支气管或小气道上皮细胞为组成性表达。IL-33与其受体ST2结合,活化核因子(NF)-κB和丝裂原激活的蛋白激酶(MAPK),诱导辅助性T细胞(Th)2细胞因子的产生,在多种炎症与免疫过程中发挥重要作用。目前,关于IL-33功能的研究在国内外都尚处于发展阶段。本研究用分子生物学技术克隆小鼠IL-33基因,通过构建其真核及原核表达载体、原核表达系统表达IL-33蛋白及其兔抗多克隆抗体的制备,探讨小鼠IL-33蛋白在不同组织中的表达分布及IL-33在不同疾病动物模型中的作用,为进一步研究其功能奠定了基础。本文主要研究内容如下: 一、根据GenBank中小鼠IL-33基因序列(No. AY905582)设计特异性引物,用逆转录-多聚酶链反应(RT-PCR)技术,克隆出IL-33基因的全长编码区。将其克隆入pcDNA3.1(+)载体,构建其真核表达载体pcDNA3.1(+)-mIL-33,之后将该表达载体转化入大肠杆菌TOP10中,经菌落PCR、酶切鉴定及序列测序,得知它与GenBank中的序列完全一致,为IL-33基因的表达研究奠定了基础。二、根据小鼠IL-33基因序列设计特异性引物,用PCR技术,克隆出IL-33成熟蛋白编码区,将其克隆入载体pET-44,构建原核表达载体pET-44-mIL-33,经测序鉴定其编码基因与GenBank中的序列一致。将该表达载体转化入大肠杆菌BL21(DE3)中获得表达菌株,然后在25°C用IPTG诱导,表达的融合蛋白以可溶性蛋白及包涵体形式存在。用镍离子亲和层析法纯化以可溶性形式存在的目的蛋白,经凝胶图像分析确定其纯度可高达95%以上。细胞增殖实验结果表明,重组IL-33蛋白具有抑制细胞增殖的活性,且其活性比标准品高。三、利用纯化的IL-33蛋白,免疫新西兰白兔,制备兔抗小鼠IL-33多克隆抗体, ELISA结果显示,该多克隆抗体效价高达1:32 000;Western blot结果显示,该多克隆抗体可以与小鼠IL-33蛋白特异性结合。免疫组织化学染色结果显示,小鼠IL-33蛋白分布于支气管上皮细胞及肝细胞的细胞质或细胞核中。四、在IL-33与类风湿关节炎关系的研究中发现,抗IL-33多克隆抗体可减轻关节炎体征及关节腔内炎症细胞的浸润。总之,IL-33基因的克隆、成熟蛋白的原核表达、多克隆抗体的制备及对IL-33与类风湿性关节炎等炎症与免疫性疾病关系的初步探讨,为进一步进行IL-33的理论研究及临床应用研究奠定了基础。
[Abstract]:Interleukin (IL)-33 is a proinflammatory cytokine found and identified by Schmitz et al. Its gene sequence and structure are most similar to those of IL-1 尾 and IL-18, members of the IL-1 family, and belong to a new member of the IL-1 family, also known as IL-1F11.IL-33 mRNA in the stomach, lungs, spinal cord, brain, skin and stationary dendritic cells of mice. Activated macrophages are highly expressed. IL-33 is constitutively expressed in human smooth muscle cells and bronchial or small airway epithelial cells. IL-33 binds to its receptor ST2. Activation of nuclear factor (NF)-魏 B and mitogen-activated protein kinase (MAPK), induces the production of (Th) 2 cytokines in helper T cells and plays an important role in various inflammatory and immune processes. At present, the research on the function of IL-33 is still in the stage of development at home and abroad. In this study, mouse IL-33 gene was cloned by molecular biology, and its eukaryotic and prokaryotic expression vectors were constructed to express IL-33 protein and rabbit anti-polyclonal antibody in prokaryotic expression system. To study the expression and distribution of mouse IL-33 protein in different tissues and the role of IL-33 in animal models of different diseases, which laid a foundation for further study of its function. The main contents of this paper are as follows: 1. According to the mouse IL-33 gene sequence in GenBank (No. The full length coding region of IL-33 gene was cloned by reverse transcription-polymerase chain reaction (RT-PCR). The eukaryotic expression vector pcDNA3.1 () -mIL-33 was constructed by cloning it into pcDNA3.1 () vector. The expression vector was transformed into Escherichia coli TOP10. It was identified by restriction endonuclease digestion and sequenced. It lays a foundation for the study of IL-33 gene expression. Secondly, specific primers were designed according to the sequence of mouse IL-33 gene, the coding region of IL-33 mature protein was cloned by PCR technique, and cloned into the vector pET-44. the prokaryotic expression vector pET-44-mIL-33 was constructed. The sequence of the encoding gene was identical to that of GenBank. The expression vector was transformed into Escherichia coli BL21 (DE3) to obtain the expressed strain. The fusion protein was induced by IPTG at 25 掳C. the expressed fusion protein existed in the form of soluble protein and inclusion body. The soluble protein was purified by nickel ion affinity chromatography. The purity of the protein was up to 95% by gel image analysis. The results of cell proliferation test showed that the recombinant IL-33 protein had the activity of inhibiting cell proliferation, and its activity was higher than that of the standard. Third, the purified IL-33 protein was used to immunize New Zealand white rabbits to prepare rabbit anti-mouse IL-33 polyclonal antibody. The results of ELISA showed that the polyclonal antibody titers were as high as 1:32 blot. The results showed that the polyclonal antibody could specifically bind to mouse IL-33 protein. Immunohistochemical staining showed that mouse IL-33 protein was distributed in cytoplasm or nucleus of bronchial epithelial cells and hepatocytes. 4. In the study of the relationship between IL-33 and rheumatoid arthritis, it was found that anti IL-33 polyclonal antibody can reduce the arthritis signs and inflammatory cell infiltration in articular cavity. In a word, the cloning of IL-33 gene, prokaryotic expression of mature protein, preparation of polyclonal antibody and preliminary study on the relationship between IL-33 and inflammation and immune diseases, such as rheumatoid arthritis, etc. It lays a foundation for further theoretical research and clinical application of IL-33.
【学位授予单位】：汕头大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392.1

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