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  本文選題：甘露糖受體 + 樹突狀細(xì)胞��； 參考：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2005年碩士論文

【摘要】：以T淋巴細(xì)胞為主的細(xì)胞免疫應(yīng)答在機(jī)體的抗腫瘤機(jī)制中起著至關(guān)重要的作用。在產(chǎn)生細(xì)胞免疫應(yīng)答的過程中,抗原的特性和樹突狀細(xì)胞(DC)的功能狀態(tài)是抗原呈遞的關(guān)鍵。隨著DC的成熟,DC誘導(dǎo)T淋巴細(xì)胞應(yīng)答的能力亦增強(qiáng)。目前,基于DC的腫瘤疫苗研究是腫瘤免疫治療研究領(lǐng)域的熱點(diǎn)。 原癌基因HER-2/neu的產(chǎn)物在人類多種上皮來源的腫瘤細(xì)胞中異常過表達(dá),它參與了腫瘤的發(fā)生、發(fā)展及惡性化轉(zhuǎn)移,而在正常組織和細(xì)胞中,HER-2/neu則低表達(dá)或不表達(dá)。因此,HER-2/neu是免疫治療的良好靶分子。HER-2/neu配體結(jié)合區(qū)第2結(jié)構(gòu)域(RLD2)是HER-2/neu蛋白的胞外區(qū)結(jié)構(gòu)域。在本研究中,根據(jù)DC表面甘露糖受體可介導(dǎo)高效的抗原內(nèi)化,我們表達(dá)、純化了HER-2/neu RLD2蛋白,并對其進(jìn)行了甘露糖修飾,以增強(qiáng)DC對抗原的攝取、呈遞,促進(jìn)DC成熟,進(jìn)一步增強(qiáng)針對抗原的細(xì)胞免疫應(yīng)答,從而為新型甘露糖化腫瘤疫苗的研制提供理論依據(jù)。 本課題研究的主要內(nèi)容包括: 第一部分 RLD2蛋白表達(dá)及純化 采用本室已構(gòu)建的重組質(zhì)粒p7IG-TrxA/RLD2轉(zhuǎn)化感受態(tài)BL21(DE3),獲得陽性轉(zhuǎn)化克隆,并獲得TrxA與RLD2蛋白在大腸桿菌中的可溶性共表達(dá)。表達(dá)產(chǎn)物經(jīng)免疫印記分析可被抗HER-2/neu特異性抗體識別。經(jīng)離子交換層析和鎳親和層析純化,RLD2蛋白的純度達(dá)85%。用質(zhì)譜法分析RLD2蛋白的分子量,與預(yù)期大小相符。HER-2/neu RLD2蛋白的高效可溶性表達(dá)為蛋白抗原的甘露糖化修飾打下了基礎(chǔ)。 第二部分 RLD2蛋白的糖基化修飾和熒光素標(biāo)記 采用化學(xué)方法對純化的RLD2蛋白(L2)進(jìn)行了甘露糖化修飾,通過質(zhì)譜法對糖基化產(chǎn)物進(jìn)行分析。結(jié)果顯示,RLD2擬糖蛋白(mL2)在質(zhì)譜圖上呈現(xiàn)甘露糖修飾蛋白的分子量預(yù)期峰形。隨后對修飾與未修飾的蛋白用FITC標(biāo)記,以利于下一步甘露糖化受體介導(dǎo)的DC抗原攝取和提呈的實(shí)驗(yàn)研究。 第三部分 人外周血來源DC的誘導(dǎo) 從健康志愿者的外周血中分離單個核細(xì)胞,經(jīng)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.
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2005
【分類號】：R392

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本文編號：1919151