【摘要】：白細(xì)胞介素(IL)-33是2005年Schmitz等使用計(jì)算機(jī)序列分析、發(fā)現(xiàn)并鑒定的一種前炎癥細(xì)胞因子。它的基因序列和結(jié)構(gòu)與IL-1家族成員IL-1β和IL-18最為相似,屬于IL-1家族的一個(gè)新成員,也被稱(chēng)為IL-1F11。IL-33 mRNA在小鼠胃、肺、脊髓、腦、皮膚等組織及靜止樹(shù)突狀細(xì)胞、活化的巨噬細(xì)胞高表達(dá),在人平滑肌細(xì)胞及支氣管或小氣道上皮細(xì)胞為組成性表達(dá)。IL-33與其受體ST2結(jié)合,活化核因子(NF)-κB和絲裂原激活的蛋白激酶(MAPK),誘導(dǎo)輔助性T細(xì)胞(Th)2細(xì)胞因子的產(chǎn)生,在多種炎癥與免疫過(guò)程中發(fā)揮重要作用。 目前,關(guān)于IL-33功能的研究在國(guó)內(nèi)外都尚處于發(fā)展階段。本研究用分子生物學(xué)技術(shù)克隆小鼠IL-33基因,通過(guò)構(gòu)建其真核及原核表達(dá)載體、原核表達(dá)系統(tǒng)表達(dá)IL-33蛋白及其兔抗多克隆抗體的制備,探討小鼠IL-33蛋白在不同組織中的表達(dá)分布及IL-33在不同疾病動(dòng)物模型中的作用,為進(jìn)一步研究其功能奠定了基礎(chǔ)。本文主要研究?jī)?nèi)容如下: 一、根據(jù)GenBank中小鼠IL-33基因序列(No. AY905582)設(shè)計(jì)特異性引物,用逆轉(zhuǎn)錄-多聚酶鏈反應(yīng)(RT-PCR)技術(shù),克隆出IL-33基因的全長(zhǎng)編碼區(qū)。將其克隆入pcDNA3.1(+)載體,構(gòu)建其真核表達(dá)載體pcDNA3.1(+)-mIL-33,之后將該表達(dá)載體轉(zhuǎn)化入大腸桿菌TOP10中,經(jīng)菌落PCR、酶切鑒定及序列測(cè)序,得知它與GenBank中的序列完全一致,為IL-33基因的表達(dá)研究奠定了基礎(chǔ)。 二、根據(jù)小鼠IL-33基因序列設(shè)計(jì)特異性引物,用PCR技術(shù),克隆出IL-33成熟蛋白編碼區(qū),將其克隆入載體pET-44,構(gòu)建原核表達(dá)載體pET-44-mIL-33,經(jīng)測(cè)序鑒定其編碼基因與GenBank中的序列一致。將該表達(dá)載體轉(zhuǎn)化入大腸桿菌BL21(DE3)中獲得表達(dá)菌株,然后在25°C用IPTG誘導(dǎo),表達(dá)的融合蛋白以可溶性蛋白及包涵體形式存在。用鎳離子親和層析法純化以可溶性形式存在的目的蛋白,經(jīng)凝膠圖像分析確定其純度可高達(dá)95%以上。細(xì)胞增殖實(shí)驗(yàn)結(jié)果表明,重組IL-33蛋白具有抑制細(xì)胞增殖的活性,且其活性比標(biāo)準(zhǔn)品高。 三、利用純化的IL-33蛋白,免疫新西蘭白兔,制備兔抗小鼠IL-33多克隆抗體, ELISA結(jié)果顯示,該多克隆抗體效價(jià)高達(dá)1:32 000;Western blot結(jié)果顯示,該多克隆抗體可以與小鼠IL-33蛋白特異性結(jié)合。免疫組織化學(xué)染色結(jié)果顯示,小鼠IL-33蛋白分布于支氣管上皮細(xì)胞及肝細(xì)胞的細(xì)胞質(zhì)或細(xì)胞核中。 四、在IL-33與類(lèi)風(fēng)濕關(guān)節(jié)炎關(guān)系的研究中發(fā)現(xiàn),抗IL-33多克隆抗體可減輕關(guān)節(jié)炎體征及關(guān)節(jié)腔內(nèi)炎癥細(xì)胞的浸潤(rùn)。 總之,IL-33基因的克隆、成熟蛋白的原核表達(dá)、多克隆抗體的制備及對(duì)IL-33與類(lèi)風(fēng)濕性關(guān)節(jié)炎等炎癥與免疫性疾病關(guān)系的初步探討,為進(jìn)一步進(jìn)行IL-33的理論研究及臨床應(yīng)用研究奠定了基礎(chǔ)。
[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 擄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.
【學(xué)位授予單位】：汕頭大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類(lèi)號(hào)】：R392.1

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