β-淀粉样多肽人源性单链抗体的制备与功能鉴定
发布时间：2018-10-23 15:24
【摘要】：阿尔茨海默病(AD)患者脑内β-淀粉样多肽(Aβ)的形成和积聚，具有直接和间接的神经毒性作用，在AD发病过程中起关键作用。阻止AD患者脑内Aβ积聚并予以清除已成为抗AD病因学疗法的重要靶点。靶向Aβ的免疫治疗，特异性高，副作用小。抗Aβ抗体通过多种机制介导脑内Aβ的清除，减轻转基因AD模型小鼠脑内的Aβ负荷，缓解认知障碍。然而单克隆抗体的鼠源性特征限制其在临床使用。本研究试图从全人源性噬菌体抗体库中筛选出抗Aβ特异性单链抗体，在充分鉴定其生物学活性及其可能的抗Aβ药理学作用后，建立新的AD免疫疗法。 研究中，首先以Aβ_(1-40)为靶标，应用噬菌体抗体库技术，，结果经过五轮“吸附-洗脱-扩增”亲和筛选，随机挑取单克隆噬菌体抗体进行噬菌体ELISA检测，结果发现一株高特异性抗Aβ噬菌体抗体——E3克隆。进一步提取E3克隆噬菌粒，转化HB2151诱导表达，产生抗Aβ可溶性E3单链抗体，并进行His-trap亲和层析纯化。Westernblot证实E3单链抗体可与人工合成的Aβ特异性结合，免疫组织化学结果显示，E3单链抗体还可与大脑皮层神经元天然分泌的Aβ特异性结合。将E3单链抗体与Aβ单体或Aβ纤维在体外共同孵育，透射电子显微镜和硫黄素T荧光分析显示，E3单链抗体在体外不仅可阻止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.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392
本文编号：2289651
[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.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392
【参考文献】
相关期刊论文 前1条
1 杨志勇,汪华侨,张革,林贤,谢瑶,袁群芳,姚志彬;国人外周血抗β-淀粉样蛋白抗体水平的依龄性变化[J];中山大学学报(医学科学版);2005年02期
本文编号：2289651
本文链接：https://www.wllwen.com/yixuelunwen/binglixuelunwen/2289651.html
最近更新
教材专著
