本文關(guān)鍵詞： 阿爾茨海默病 β-淀粉樣多肽 單克隆抗體 嵌合抗體 Morris水迷宮 抗體人源化改造　出處：《中國協(xié)和醫(yī)科大學(xué)》2007年碩士論文　論文類型：學(xué)位論文

【摘要】： 阿爾茨海默病(Alzheimer's disease，AD)是中樞神經(jīng)系統(tǒng)一種常見的進(jìn)行性神經(jīng)退行性變疾病，也是癡呆的最常見類型，其典型病理學(xué)特征包括：淀粉樣斑塊(即老年斑Senior Plaque，SP)、神經(jīng)纖維纏結(jié)(neurofibrillary tangles，NFT)和神經(jīng)突觸減少或神經(jīng)元丟失。目前國內(nèi)外還沒有針對AD治療的特效藥問世。β—淀粉樣多肽(β-amyloid peptide，Aβ)被認(rèn)為是導(dǎo)致神經(jīng)元損傷及認(rèn)知記憶功能衰退的主要致病物質(zhì)。大量動物試驗(yàn)研究表明，針對Aβ的免疫療法(主動免疫或被動免疫)可以有效減少APP轉(zhuǎn)基因鼠腦內(nèi)聚集的Aβ纖維，而且能夠明顯改善AD模型鼠的認(rèn)知狀況�？笰β抗體研究為AD治療提供了新思路，可能成為AD治療的新途徑。 本課題基于抗Aβ抗體治療AD的新思路，研究基因工程制備抗Aβ抗體及其人源化改造的可能性。主要包括三個部分：抗Aβ_(1-42)單克隆抗體制備，抗Aβ單抗對AD模型動物治療效果和該單抗人源化改造初步研究。 一．抗Aβ_(1-42)單克隆抗體制備及其性質(zhì)鑒定 Aβ_(1-42)是4kDa左右的小分子多肽，免疫原性較弱，制備單抗比較困難。開始時采用常規(guī)制備單抗方法操作，結(jié)果僅僅獲得IgM型單克隆抗體，其效價不高且純化比較困難，經(jīng)過改進(jìn)免疫方法，提高了小分子多肽的免疫效果，最終得到了IgG型抗Aβ_(1-42)的單抗。進(jìn)行單抗的性質(zhì)鑒定，抗體為IgG1亞型，識別Aβ表位的范圍為AβN端序列13-28位氨基酸。其腹水效價達(dá)1：1×10~6、親和力達(dá)到1×10~9 M(-1)水平，經(jīng)Protein A親和層析柱純化抗體，純度可達(dá)90％以上。 二．抗Aβ_(1-42)單克隆抗體對癡呆動物治療作用 國外有研究證明抗Aβ單抗對AD模型動物有一定治療效果，而國內(nèi)還沒有相關(guān)的報道。為驗(yàn)證獲得的單抗對AD的治療作用，我們進(jìn)行了AD模型鼠的體內(nèi)試驗(yàn)。將BALB／c鼠分為四組：陽性對照(模型鼠)組、空白對照(正常小鼠)組、單抗治療組及假手術(shù)組。通過在小鼠海馬區(qū)一次性注射10μg Aβ來構(gòu)建AD鼠模型，單抗治療組在建模注射Aβ前三天開始行腹腔注射抗Aβ單抗(500μg／只)，每隔三天注射一次，共給藥四次，最后一次注射在建模后第八天。建模注射Aβ后第12天開始進(jìn)行Morris水迷宮行為學(xué)試驗(yàn)，連續(xù)進(jìn)行三天，，之后處死小鼠取腦、固定。經(jīng)統(tǒng)計學(xué)分析結(jié)果顯示，陽性對照(模型鼠)組在Morris水迷宮行為學(xué)試驗(yàn)中，平均尋找隱性平臺潛伏期明顯延長，與空白對照組比較有顯著差異(P＜0.05)。單抗治療組與陽性對照組比較尋找隱性平臺潛伏期明顯縮短(P＜0.05)。行為學(xué)評價結(jié)果顯示，抗Aβ單抗在體內(nèi)能夠改善由Aβ腦內(nèi)注射引起的學(xué)習(xí)記憶損傷。 三．抗Aβ_(1-42)單克隆抗體的人源化改造研究 鼠源單克隆抗體用于人體前，需要進(jìn)行抗體人源化改造。本課題單抗人源化改造研究的階段性工作為構(gòu)建人-鼠IgG嵌合抗體基因表達(dá)體系，方法為先分別收集雜交瘤細(xì)胞(10~7左右)，Trizol法提取細(xì)胞總RNA，然后用美國BD公司的逆轉(zhuǎn)錄試劑盒進(jìn)行RT-PCR獲得cDNA第一條鏈，再設(shè)計特異引物分別擴(kuò)增單抗輕鏈和重鏈的可變區(qū)基因，并利用重組PCR(Overlap PCR)方法分別將鼠源單抗輕重鏈可變區(qū)基因與人源單抗輕重鏈恒定區(qū)基因連接構(gòu)建嵌合基因，然后克隆到pcDNA3.1真核表達(dá)載體內(nèi)，轉(zhuǎn)染哺乳動物細(xì)胞，表達(dá)基因工程抗體。我們成功獲得了輕鏈和重鏈的人-鼠嵌合抗體基因，并克隆入真核表達(dá)載體。本課題成功構(gòu)建單抗嵌合基因?yàn)橐院髥慰惯M(jìn)一步人源化改造研究奠定了基礎(chǔ)。 本課題中我們成功制備并獲得了抗Aβ1-42的IgG1型單克隆抗體，該抗體能夠特異識別Aβ肽段并與之結(jié)合，腹水效價達(dá)1：1×10~6，親和力達(dá)1×10~9M~(-1)。動物試驗(yàn)研究證明其能夠改善AD模型動物的認(rèn)知記憶功能。同時我們還克隆得到該抗體的基因，得到了其可變區(qū)序列信息，并成功構(gòu)建人-鼠嵌合抗體基因，為以后單抗的進(jìn)一步人源化改造奠定了基礎(chǔ)。我們獲得的單克隆抗體可以為AD治療的研究提供有力的工具，但其改善AD模型動物的認(rèn)知記憶功能的機(jī)制及能否應(yīng)用于臨床研究，還需要進(jìn)一步探討。
[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.

【學(xué)位授予單位】：中國協(xié)和醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R392

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