蛋白质折叠是蛋白质形成正确空间结构并执行生物学功能重要的一步。而蛋白质由于各种各样的因素导致无法正确折叠，就有可能造成蛋白质高级结构的错误，以及功能异常，从而导致各种疾病，这些疾病即为蛋白质折叠病。而神经退行性疾病阿尔兹海默症就是其中的一种。

阿尔兹海默症的病理性特症之一是神经纤维缠结，而这是由于Tau蛋白异常磷酸化形成错误聚集体导致的。因此通过研究有哪些因素会影响Tau蛋白的聚集以及聚集的机制，并以此来减少聚集体的形成，可以为寻找预防或减缓阿尔兹海默症的治疗方法打下基础。单链抗体分子由于具有特异性高、分子量小、可透过血脑屏障等特点，成为影响Tau聚集及其细胞毒性的一种重要因子。除此之外，在生物体内存在的渗透剂分子对于促使蛋白质正确折叠，辅助蛋白质形成有活性的空间结构也有作用，也可能对Tau蛋白的聚集产生影响。生物大分子的液-液相分离现象近来被发现对于各种生命活动都具有重要的调节作用，且液-液相分离的失调被认为是多种神经退行性疾病的发病原因，探究Tau蛋白聚集过程的液-液相分离，对于揭示Tau蛋白质聚集的分子机制有一定意义。

本论文运用了 Western Blot 技术、ELISA 技术研究了单链抗体 scFv A1与 磷酸化Tau蛋白（pTau40）的相互作用，并利用 ThT 荧光光谱技术检测了单链抗体scFv A1 对 pTau40 聚集的影响作用，发现scFv A1与pTau40共同孵育时，pTau40蛋白聚集程度显著下降，而且随着scFv A1浓度增大，其抑制pTau40聚集的效应也有所增强。此外还利用非变性电泳技术进一步确定scFv A1 对 pTau40 聚集的影响，发现在scFv A1的作用下，pTau40蛋白寡聚体的存在状态发生改变，其聚集被抑制，pTau40蛋白单体增多。

本论文就scFv A1对pTau40 细胞毒性的影响作用进行了初步探究，胞外孵育结合MTT及LDH实验检测结果显示：pTau40具有细胞毒性，可导致 SH-SY5Y 细胞增殖受到抑制，使细胞逐渐变圆，突触减少。scFv A1 的加入可显著削弱 pTau40 的细胞毒性，且随 scFv A1 浓度增大，其抑制作用也有所增强，scFv A1 本身未表现出细胞毒性。

本论文还利用ThT荧光技术研究了多种渗透剂分子对pTau40聚集的影响作用，发现甜菜碱分子可对pTau40的聚集产生一定的抑制作用。

本论文对pTau40的液-液相分离进行了研究，利用显微镜观测发现，在拥挤环境中，pTau40可发生显著的液-液相分离，该过程与pTau40的聚集存在一定的联系，单链抗体scFv A1及渗透剂海藻糖分子对此液-液相分离过程有一定的抑制作用。

上述实验工作为后续研究pTau40的聚集机制以及单链抗体的作用机制提供了支撑，并为预防和治疗相关蛋白折叠疾病提供了了一定的理论基础。

Protein folding is an important step for proteins to form the correct spatial structure and perform biological functions. The protein cannot be folded properly due to various factors, which may cause errors in the high-level structure of the protein and abnormal functions, leading to various diseases. These diseases are protein folding diseases. The neurodegenerative disease Alzheimer's is one of them.

One of the pathological characteristics of Alzheimer's disease is neurofibrillary tangles, which are caused by abnormal phosphorylation of Tau protein to form incorrect aggregates. Therefore, by studying the factors which can affect the aggregation of Tau protein and exploring the mechanism of aggregation, thereby reducing the formation of aggregates,we can lay the foundation for finding treatments to prevent or slow Alzheimer's disease. Single-chain antibody molecules have high specificity, small molecular weight, and can penetrate the blood-brain barrier, which has become an important factor affecting Tau aggregation and its cytotoxicity. In addition,  osmolyte present in the organism also play a role in promoting the correct folding of proteins, assisting in the formation of active spatial structures, and may also affect the aggregation of Tau proteins. The liquid-liquid phase separation of biological macromolecules has recently been found to have important regulatory effects on various life activities, and the imbalance of liquid-liquid phase separation is considered to be the cause of various neurodegenerative diseases.Exploring the liquid-liquid phase separation during the process of Tau protein aggregation has certain significance for revealing the molecular mechanism of Tau protein aggregation.

In this paper, the interaction between the scFv A1 single-chain antibody and phosphorylated Tau protein (pTau40) was studied using Western Blot technology and ELISA technology, and the effect of single-chain antibody scFv A1 on the aggregation of pTau40 was detected using ThT fluorescence spectroscopy. When scFv A1 and pTau40 were co-incubated, the degree of aggregation of pTau40 protein decreased significantly, and as scFv A1 concentration increased, its effect of inhibiting pTau40 aggregation also increased. In addition, the effect of scFv A1 on the aggregation of pTau40 was further determined using non-denaturing electrophoresis technology. It was found that under the action of scFv A1, the presence of pTau40 protein oligomers changed, its aggregation was inhibited, and pTau40 protein monomers increased.

This paper conducted a preliminary study on the effect of scFv A1 on pTau40 

cytotoxicity. The results of extracellular incubation combined with MTT and LDH experiments showed that pTau40 has cytotoxicity, which can inhibit the proliferation of SH-SY5Y cells,gradually round the cells and reduced synapses. The addition of scFv A1 can significantly weaken the cytotoxicity of pTau40, and its inhibitory effect is enhanced as the concentration of scFv A1 increases. scFv A1 itself does not show cytotoxicity.

In this paper, the effect of various osmolyte on the aggregation of pTau40 was also studied using ThT fluorescence technology, and it was found that betaine molecules can inhibit the aggregation of pTau40 to a certain extent.

In this thesis, the liquid-liquid phase separation of pTau40 was studied. Microscopic observations revealed that in a crowded environment, pTau40 can undergo significant liquid-liquid phase separation. This process is related to the aggregation of pTau40. Single-chain antibody scFv A1 and osmolyte trehalose molecules have a certain inhibitory effect on this liquid-liquid phase separation process.

The above experimental work provides support for subsequent research on the aggregation mechanism of pTau40 and the action mechanism of single chain antibodies, and provides a certain theoretical basis for the prevention and treatment of related protein folding diseases.