抗Aβ单抗的性质、生物学效应及人源化改造研究

发布时间：2018-01-19 00:21

本文关键词： 阿尔茨海默病 β-淀粉样多肽单克隆抗体嵌合抗体 Morris水迷宫抗体人源化改造　出处：《中国协和医科大学》2007年硕士论文　论文类型：学位论文

【摘要】： 阿尔茨海默病(Alzheimer's disease，AD)是中枢神经系统一种常见的进行性神经退行性变疾病，也是痴呆的最常见类型，其典型病理学特征包括：淀粉样斑块(即老年斑Senior Plaque，SP)、神经纤维缠结(neurofibrillary tangles，NFT)和神经突触减少或神经元丢失。目前国内外还没有针对AD治疗的特效药问世。β—淀粉样多肽(β-amyloid peptide，Aβ)被认为是导致神经元损伤及认知记忆功能衰退的主要致病物质。大量动物试验研究表明，针对Aβ的免疫疗法(主动免疫或被动免疫)可以有效减少APP转基因鼠脑内聚集的Aβ纤维，而且能够明显改善AD模型鼠的认知状况。抗Aβ抗体研究为AD治疗提供了新思路，可能成为AD治疗的新途径。本课题基于抗Aβ抗体治疗AD的新思路，研究基因工程制备抗Aβ抗体及其人源化改造的可能性。主要包括三个部分：抗Aβ_(1-42)单克隆抗体制备，抗Aβ单抗对AD模型动物治疗效果和该单抗人源化改造初步研究。一．抗Aβ_(1-42)单克隆抗体制备及其性质鉴定 Aβ_(1-42)是4kDa左右的小分子多肽，免疫原性较弱，制备单抗比较困难。开始时采用常规制备单抗方法操作，结果仅仅获得IgM型单克隆抗体，其效价不高且纯化比较困难，经过改进免疫方法，提高了小分子多肽的免疫效果，最终得到了IgG型抗Aβ_(1-42)的单抗。进行单抗的性质鉴定，抗体为IgG1亚型，识别Aβ表位的范围为AβN端序列13-28位氨基酸。其腹水效价达1：1×10~6、亲和力达到1×10~9 M(-1)水平，经Protein A亲和层析柱纯化抗体，纯度可达90％以上。二．抗Aβ_(1-42)单克隆抗体对痴呆动物治疗作用国外有研究证明抗Aβ单抗对AD模型动物有一定治疗效果，而国内还没有相关的报道。为验证获得的单抗对AD的治疗作用，我们进行了AD模型鼠的体内试验。将BALB／c鼠分为四组：阳性对照(模型鼠)组、空白对照(正常小鼠)组、单抗治疗组及假手术组。通过在小鼠海马区一次性注射10μg Aβ来构建AD鼠模型，单抗治疗组在建模注射Aβ前三天开始行腹腔注射抗Aβ单抗(500μg／只)，每隔三天注射一次，共给药四次，最后一次注射在建模后第八天。建模注射Aβ后第12天开始进行Morris水迷宫行为学试验，连续进行三天，，之后处死小鼠取脑、固定。经统计学分析结果显示，阳性对照(模型鼠)组在Morris水迷宫行为学试验中，平均寻找隐性平台潜伏期明显延长，与空白对照组比较有显著差异(P＜0.05)。单抗治疗组与阳性对照组比较寻找隐性平台潜伏期明显缩短(P＜0.05)。行为学评价结果显示，抗Aβ单抗在体内能够改善由Aβ脑内注射引起的学习记忆损伤。三．抗Aβ_(1-42)单克隆抗体的人源化改造研究鼠源单克隆抗体用于人体前，需要进行抗体人源化改造。本课题单抗人源化改造研究的阶段性工作为构建人-鼠IgG嵌合抗体基因表达体系，方法为先分别收集杂交瘤细胞(10~7左右)，Trizol法提取细胞总RNA，然后用美国BD公司的逆转录试剂盒进行RT-PCR获得cDNA第一条链，再设计特异引物分别扩增单抗轻链和重链的可变区基因，并利用重组PCR(Overlap PCR)方法分别将鼠源单抗轻重链可变区基因与人源单抗轻重链恒定区基因连接构建嵌合基因，然后克隆到pcDNA3.1真核表达载体内，转染哺乳动物细胞，表达基因工程抗体。我们成功获得了轻链和重链的人-鼠嵌合抗体基因，并克隆入真核表达载体。本课题成功构建单抗嵌合基因为以后单抗进一步人源化改造研究奠定了基础。本课题中我们成功制备并获得了抗Aβ1-42的IgG1型单克隆抗体，该抗体能够特异识别Aβ肽段并与之结合，腹水效价达1：1×10~6，亲和力达1×10~9M~(-1)。动物试验研究证明其能够改善AD模型动物的认知记忆功能。同时我们还克隆得到该抗体的基因，得到了其可变区序列信息，并成功构建人-鼠嵌合抗体基因，为以后单抗的进一步人源化改造奠定了基础。我们获得的单克隆抗体可以为AD治疗的研究提供有力的工具，但其改善AD模型动物的认知记忆功能的机制及能否应用于临床研究，还需要进一步探讨。
[Abstract]:Alzheimer's disease (Alzheimer's disease AD) is the central nervous system is a common progressive neurodegenerative disease, is the most common type of dementia, the typical pathological features including: amyloid plaques (i.e. senile plaques Senior Plaque, SP), neurofibrillary tangles (neurofibrillary, tangles, NFT) and synapses reduce or neuronal loss. At present there is no specific treatment for AD. The advent of beta amyloid polypeptide (beta -amyloid, peptide, A) is thought to be the main pathogenic substances of neuronal injury and cognitive function decline. A large number of animal experiments show that for the immunotherapy of A beta (active or passive immunization Immune) can effectively reduce A beta fiber aggregation of APP transgenic mice in the brain, and can significantly improve the cognitive status of AD rats. The study of antibodies against A and provides a new idea for the treatment of AD, may be AD A new approach to treatment.
The new idea of antibodies against A in the treatment of AD based on the possibility of gene engineering preparation of antibodies against A and hunamnization. Mainly includes three parts: Anti A beta _ (1-42) monoclonal antibody, anti A monoclonal antibody and preliminary study on beta modification in animal models of AD and the therapeutic effect humanized monoclonal antibody.
A _. Anti A beta (1-42) monoclonal antibody preparation and characterization
A beta _ (1-42) is a low molecular weight polypeptide of about 4kDa, weak immunogenicity, monoclonal antibody preparation is difficult. At the beginning of the routine preparation of monoclonal antibody method, results only type IgM monoclonal antibody, its potency is not high and the purification difficult, improved immunity method, improve the immune effect of small molecules polypeptide, obtained IgG anti A beta _ (1-42) mAb. Characterization of monoclonal antibody, antibody of IgG1 subtype, A epitope identification of beta A beta N terminal sequence of 13-28 amino acids. The titer of ascites was 1:1 * 10~6, affinity reached 1 * 10~9 (-1) M the level of Protein by A affinity purified antibody affinity chromatography, the purity of more than 90%.
Two. Anti A beta _ (1-42) effect of monoclonal antibody on the treatment of dementia animal
Foreign studies have shown that anti A beta monoclonal antibody has a certain therapeutic effect in animal models of AD, and there are no relevant reports. In order to verify the therapeutic effect of monoclonal antibody on AD, we performed in vivo AD model rats. BALB / C were divided into four groups: positive control group (rats). The blank control group (normal mice), monoclonal antibody treatment group and sham operation group. To construct AD rat model in mice hippocampus injected 10 g A beta, monoclonal antibody treatment group by intraperitoneal injection of anti A monoclonal antibody in beta three days before modeling of injection of A beta (500 g / only), every three days injection time, were administered four times, the last time in eighth days after the injection of modeling. Modeling of injection of A beta twelfth days after the Morris water maze test, for three consecutive days, the mice were killed after the brain was removed after fixation. The results of statistical analysis showed that the positive control group (rats) Morris water maze for For the test, the average looking for hidden platform was significantly prolonged, had a significant difference compared with the blank control group (P < 0.05). Monoclonal antibody treatment group and positive control group for the hidden platform was significantly shortened (P < 0.05). Behavioral evaluation results showed that the anti A monoclonal antibody can improve beta learning and memory impairment induced by injecting by A beta in the brain in vivo.
Three. Anti A beta _ (1-42) study of humanized monoclonal antibody
The murine monoclonal antibody to human body, the need for antibody humanization. The stage work of this topic research humanized monoclonal antibody expression system for the construction of human mouse chimeric antibody IgG gene, were collected as the first method of hybridoma cells (10~7), Trizol cell total RNA extraction method, then by reverse transcriptase the kit of BD company RT-PCR to get the first cDNA chain variable region gene specific primers were amplified and then monoclonal antibody light chain and heavy chain, and the use of recombinant PCR (Overlap PCR) methods respectively, gene and human monoclonal antibody VH monoclonal antibody light chain constant region to construct chimeric gene connection. And then cloned into the pcDNA3.1 eukaryotic expression vector, transfection of mammalian cells, the expression of gene engineering antibody. We successfully obtained the chimeric antibody light chain and heavy chain of human mouse gene was cloned into the eukaryotic expression vector. The successful construction of the mAb chimeric base has laid the foundation for the further study of the further human transformation of McAbs.
In this paper we successfully prepared and obtained IgG1 monoclonal antibody against A beta 1-42, the antibody can specifically recognize A beta peptide and combined with ascites titer was 1:1 * 10~6, 1 * 10~9M~ affinity (-1). Animal experiment proved that the cognitive and memory function which can improve the animal model of AD at the same time. We also cloned the antibody genes, the variable region sequence information, and the chimeric antibody gene was successfully constructed, which lays a foundation for the further humanized monoclonal antibody. Then provide a powerful tool to study we obtained a monoclonal antibody for AD treatment, but the mechanism of cognitive and memory function improvement of AD animal model and can be applied to clinical research, also need to be further explored.

【学位授予单位】：中国协和医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

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