【摘要】：阿爾茨海默病(AD)患者腦內(nèi)β-淀粉樣多肽(Aβ)的形成和積聚，具有直接和間接的神經(jīng)毒性作用，在AD發(fā)病過程中起關(guān)鍵作用。阻止AD患者腦內(nèi)Aβ積聚并予以清除已成為抗AD病因?qū)W療法的重要靶點。靶向Aβ的免疫治療，特異性高，副作用小。抗Aβ抗體通過多種機制介導(dǎo)腦內(nèi)Aβ的清除，減輕轉(zhuǎn)基因AD模型小鼠腦內(nèi)的Aβ負(fù)荷，緩解認(rèn)知障礙。然而單克隆抗體的鼠源性特征限制其在臨床使用。本研究試圖從全人源性噬菌體抗體庫中篩選出抗Aβ特異性單鏈抗體，在充分鑒定其生物學(xué)活性及其可能的抗Aβ藥理學(xué)作用后，建立新的AD免疫療法。 研究中，首先以Aβ_(1-40)為靶標(biāo)，應(yīng)用噬菌體抗體庫技術(shù)，，結(jié)果經(jīng)過五輪“吸附-洗脫-擴增”親和篩選，隨機挑取單克隆噬菌體抗體進行噬菌體ELISA檢測，結(jié)果發(fā)現(xiàn)一株高特異性抗Aβ噬菌體抗體——E3克隆。進一步提取E3克隆噬菌粒，轉(zhuǎn)化HB2151誘導(dǎo)表達，產(chǎn)生抗Aβ可溶性E3單鏈抗體，并進行His-trap親和層析純化。Westernblot證實E3單鏈抗體可與人工合成的Aβ特異性結(jié)合，免疫組織化學(xué)結(jié)果顯示，E3單鏈抗體還可與大腦皮層神經(jīng)元天然分泌的Aβ特異性結(jié)合。將E3單鏈抗體與Aβ單體或Aβ纖維在體外共同孵育，透射電子顯微鏡和硫黃素T熒光分析顯示，E3單鏈抗體在體外不僅可阻止Aβ
[Abstract]:The formation and accumulation of 尾 -amyloid polypeptide (A 尾) in the brain of (AD) patients with Alzheimer's disease have direct and indirect neurotoxic effects and play a key role in the pathogenesis of AD. Preventing the accumulation and clearance of A 尾 in the brain of AD patients has become an important target of anti-AD etiology therapy. The immunotherapy targeting A 尾 has high specificity and little side effect. Anti-A 尾 antibody mediates the clearance of A 尾 in the brain through various mechanisms, reduces the A 尾 load in the brain of transgenic AD model mice, and alleviates cognitive impairment. However, the murine characteristics of monoclonal antibodies limit their clinical use. In this study, we try to screen the anti-A 尾 specific scFv from the whole human phage antibody library. After fully identifying its biological activity and its possible anti-A 尾 pharmacological effect, a new AD immunotherapy was established. In this study, A 尾 _ (1-40) was used as the target and phage antibody library technique was used. The results showed that after five rounds of "adsorption-eluation-amplification" affinity screening, monoclonal phage antibodies were randomly selected for phage ELISA detection. Results A highly specific anti A 尾 phage antibody E 3 clone was found. E3 cloned macrophages were further extracted, transformed into HB2151 to induce expression, to produce anti-A 尾 soluble E3 scFv, and purified by His-trap affinity chromatography. Westernblot confirmed that E3 scFv could specifically bind to synthetic A 尾. Immunohistochemical results showed that E3 scFv could also bind to A 尾 secreted naturally by cortical neurons. E3 scFv was incubated with A 尾 monomer or A 尾 fiber in vitro. Transmission electron microscopy and T fluorescence analysis showed that E3 scFv could not only inhibit A 尾 in vitro.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2006
【分類號】：R392

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相關(guān)期刊論文 前1條

1 楊志勇,汪華僑,張革,林賢,謝瑤,袁群芳,姚志彬;國人外周血抗β-淀粉樣蛋白抗體水平的依齡性變化[J];中山大學(xué)學(xué)報(醫(yī)學(xué)科學(xué)版);2005年02期

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