受体介导的甘露糖化蛋白抗原的摄

发布时间：2018-05-21 12:17

本文选题：甘露糖受体 + 树突状细胞　；参考：《中国人民解放军军事医学科学院》2005年硕士论文

【摘要】：以T淋巴细胞为主的细胞免疫应答在机体的抗肿瘤机制中起着至关重要的作用。在产生细胞免疫应答的过程中,抗原的特性和树突状细胞(DC)的功能状态是抗原呈递的关键。随着DC的成熟,DC诱导T淋巴细胞应答的能力亦增强。目前,基于DC的肿瘤疫苗研究是肿瘤免疫治疗研究领域的热点。原癌基因HER-2/neu的产物在人类多种上皮来源的肿瘤细胞中异常过表达,它参与了肿瘤的发生、发展及恶性化转移,而在正常组织和细胞中,HER-2/neu则低表达或不表达。因此,HER-2/neu是免疫治疗的良好靶分子。HER-2/neu配体结合区第2结构域(RLD2)是HER-2/neu蛋白的胞外区结构域。在本研究中,根据DC表面甘露糖受体可介导高效的抗原内化,我们表达、纯化了HER-2/neu RLD2蛋白,并对其进行了甘露糖修饰,以增强DC对抗原的摄取、呈递,促进DC成熟,进一步增强针对抗原的细胞免疫应答,从而为新型甘露糖化肿瘤疫苗的研制提供理论依据。本课题研究的主要内容包括: 第一部分 RLD2蛋白表达及纯化采用本室已构建的重组质粒p7IG-TrxA/RLD2转化感受态BL21(DE3),获得阳性转化克隆,并获得TrxA与RLD2蛋白在大肠杆菌中的可溶性共表达。表达产物经免疫印记分析可被抗HER-2/neu特异性抗体识别。经离子交换层析和镍亲和层析纯化,RLD2蛋白的纯度达85%。用质谱法分析RLD2蛋白的分子量,与预期大小相符。HER-2/neu RLD2蛋白的高效可溶性表达为蛋白抗原的甘露糖化修饰打下了基础。第二部分 RLD2蛋白的糖基化修饰和荧光素标记采用化学方法对纯化的RLD2蛋白(L2)进行了甘露糖化修饰,通过质谱法对糖基化产物进行分析。结果显示,RLD2拟糖蛋白(mL2)在质谱图上呈现甘露糖修饰蛋白的分子量预期峰形。随后对修饰与未修饰的蛋白用FITC标记,以利于下一步甘露糖化受体介导的DC抗原摄取和提呈的实验研究。第三部分人外周血来源DC的诱导从健康志愿者的外周血中分离单个核细胞,经rhGM-CSF、rhIL-4及脂多糖
[Abstract]:The cellular immune response of T-lymphocytes plays an important role in the anti-tumor mechanism. In the process of producing cellular immune response, the characteristics of antigen and the functional state of dendritic cells (DC) are the key to antigen presentation. The ability of DC to induce T lymphocyte response was also increased with the maturation of DC. At present, DC-based tumor vaccine research is a hot topic in the field of tumor immunotherapy. The proto-oncogene HER-2/neu is overexpressed in various human epithelial tumor cells. It is involved in the carcinogenesis, development and malignant metastasis of human tumors, while the expression of HER-2 / neu is low or non-expressed in normal tissues and cells. Therefore, HER-2 / neu is a good target molecule for immunotherapy. The second domain of HER-2 / neu ligand binding region (RLD2) is the extracellular domain of HER-2/neu protein. In this study, HER-2/neu RLD2 protein was expressed, purified and modified with mannose to enhance the antigen uptake, presentation and maturation of DC, according to the high efficiency antigen internalization mediated by mannose receptor on DC surface. To further enhance the cellular immune response to antigen, so as to provide a theoretical basis for the development of new Mannan glycosylated tumor vaccine. The main contents of this research include: Part I expression and purification of RLD2 protein By using the recombinant plasmid p7IG-TrxA/RLD2 constructed in our laboratory, we obtained the positive transformation clone and the soluble co-expression of TrxA and RLD2 protein in Escherichia coli. The expressed product can be recognized by anti-HER-2/neu specific antibody by immunological imprinting analysis. The purity of RLD2 protein was 85% by ion exchange chromatography and nickel affinity chromatography. The molecular weight of RLD2 protein was analyzed by mass spectrometry. The high soluble expression of HER-2 / neu RLD2 protein was consistent with the expected size, which laid the foundation for the modification of the protein antigen by mannose saccharification. The second part: glycosylation modification and fluorescein labeling of RLD2 protein The purified RLD2 protein L2 was modified by mannose and the glycosylation products were analyzed by mass spectrometry. The results showed that RLD2 glycoprotein mL2) showed the expected molecular weight peak of mannose modified protein on the mass spectrogram. Then the modified and unmodified proteins were labeled with FITC to facilitate the further study on the uptake and presentation of DC antigen mediated by mannose glycosylated receptor. The third part of the induction of DC from human peripheral blood Mononuclear cells were isolated from peripheral blood of healthy volunteers. RhIL-4 and lipopolysaccharide were obtained through rhGM-CSFU rhIL-4 and lipopolysaccharide.
【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】：R392

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