大量研究表明，神经退行性疾病的主要致病原因往往是蛋白质的错误折叠和异常聚集。阿尔茨海默病作为最常见的一种人类神经退行性疾病，其最主要的病理特征之一是 Tau 蛋白的异常形式在神经元内部的积累，而这种畸变的 Tau 缠结正是由于Tau 蛋白的异常聚集导致的。

通过研究 Tau 蛋白的特性，帮助 Tau 蛋白正确折叠，可能是治疗阿尔兹海默病的有效策略。分子伴侣能够促进靶蛋白的正确折叠，据报道，有些单链抗体表现出抗原特异性的分子伴侣样活性。所以，利用噬菌体展示技术，实验室已经成功筛选得到了针对 Tau 蛋白的特异性单链抗体 A1（single-chain variable fragment antibody A1，scFv A1）。基于这些背景，本文主要探究了 Tau 蛋白的特性及其与 scFv A1 的相互作用。

本文通过活化实验室存有的菌种，在体外表达、纯化 Tau40 蛋白，并使用微管蛋白聚合测定法和透射电镜观察法鉴定了提取的 Tau40 蛋白的活性，发现大肠杆菌表达的Tau40 蛋白可以促进微管蛋白组装成微管。经糖原合酶激酶处理得到磷酸化的 Tau40（即pTau40）后，使用质谱鉴定了 pTau40 的磷酸化位点。

在体外表达、纯化 scFv A1 后，本文使用 Western Blot 验证了单链抗体 A1 与 Tau40、pTau40 都存在一定的结合能力。利用 ThT 荧光探针法和透射电镜技术探究了 scFv A1 对 pTau40 蛋白聚集的影响，发现体外 37℃ 单独孵育的 pTau40 会发生聚集，而加入 scFv A1 后，pTau40 蛋白的聚集会受到抑制。

在离体细胞水平，本文初步探究了胞外孵育的 pTau40 对于人神经母细胞瘤株 SH-SY5Y 细胞的细胞毒性以及 scFv A1对其的影响。通过增强型 CCK-8 试剂盒的检测，结果表明 pTau40 具有一定的细胞毒性，导致SH-SY5Y 细胞的形态发生了明显变化，细胞死亡率有所增加。当胞外共同孵育 pTau40 和 scFv A1时，可在一定程度上缓解 pTau40 的细胞毒性，且随 scFv A1 浓度增大，其对于毒性的缓解作用也有所增强。此外，本文根据已知的 Tau 和 scFv A1 的基因序列，成功构建了胞内的过表达载体，为后续探究 pTau40 的胞内毒性以及 scFv A1对其的影响打下了一定的实验基础。

A large number of studies have shown that the misfolding and aggregation of proteins is the main cause of neurodegenerative diseases. Alzheimer’s disease is one of the most common human neurodegenerative disease. The main pathological feature of Alzheimer’s disease is the accumulation of abnormal forms of Tau protein in neurons, and this aberrant Tau tangles is precisely caused by abnormal aggregation. Therefore, studying the properties of Tau protein to help Tau protein fold correctly may be an effective strategy for the treatment of Alzheimer's disease. Molecular chaperones can promote the correct folding of target proteins. According to reports, some single-chain antibodies may exhibit antigen-specific molecular chaperone-like activities. Therefore, using phage display technology, the laboratory has successfully screened the specific single-chain variable fragment antibody A1 (scFv A1) against Tau protein. Based on these backgrounds, this article mainly explores the characteristics of Tau protein and its interaction with scFv A1.

In this paper, Tau40 protein was expressed and purified in vitro by activating the E. coli in the laboratory, and the activity of the extracted Tau40 protein was then identified by tubulin polymerization assay and transmission electron microscopy. It was found that the Tau40 protein expressed by E. coli can promote Tubulin assembles into microtubules. After glycogen synthase kinase treatment to obtain phosphorylated Tau40 (pTau40), the phosphorylation site of pTau40 was identified by mass spectrometry.

After expressing and purifying scFv A1 in vitro, this paper used Western Blot to verify that the scFv A1 had a certain binding ability with Tau40 and pTau40. The effect of scFv A1 on pTau40 protein aggregation was explored using ThT fluorescent probe method and transmission electron microscopy technology. It was found that pTau40 that was incubated alone at 37°C in vitro would aggregate. However, when scFv A1 was added, the aggregation of pTau40 protein would be inhibited.

At the level of in vitro cells, this article preliminarily explored the cytotoxicity of pTau40 which was extracellularly incubated to human neuroblastoma line SH-SY5Y cells and the effect of scFv A1 on it. Through the detection of enhanced CCK-8 kit, the results showed that pTau40 had a certain cytotoxicity, resulting in a significant change in the morphology of SH-SY5Y cells and an increase in cell mortality. However, when pTau40 and scFv A1 were incubated together extracellularly, the cytotoxicity of pTau40 can be alleviated to a certain extent, and as the concentration of scFv A1 increases, its mitigation effect on toxicity was also enhanced. In addition, based on the known gene sequences of Tau and scFv A1, this article successfully constructed an intracellular overexpression vector, which laid a certain experimental basis for the subsequent exploration of the intracellular toxicity of pTau40 and the influence of scFv A1 on it.