抗Aβ12-35纳米抗体的制备及活性研究

发布时间：2018-07-17 17:22

【摘要】：阿尔兹海默症(Alzheimer's disease, AD)是一种严重的神经退行性疾病,其发病机制尚没有定论,很多研究表明Aβ及其聚集体是AD发生发展的重要影响因素,因此研发能够针对Aβ聚集体的抑制剂或药物成为越来越受到关注的研究热点之一。在基于抗Aβ抗体治疗AD的方法中,目前多采用人源化多克隆或单克隆抗体和小分子单链抗体等,与之相比,纳米抗体由于制备成本低、容易跨越血脑屏障等优点,在抗体疗法中受到越来越多的研究和关注。另外,已有的研究表明,Aβ的氮端不参与Aβ的聚集,用于临床实验的能够识别Aβ氮端的抗体因为能引起机体更强的炎症反应而不得不停止临床实验；而Aβ中间片段是与Aβ聚集相关的关键位点以及毒性片段,极易形成β折叠结构,而β折叠正是Aβ聚集体主要的结构特点,能够识别Aβ聚集区段的抗体的炎症反应相对较低,因此,筛选能够特异性结合Aβ中间段的纳米抗体对于抑制Aβ的聚集和探索Aβ聚集机制将具有重要意义。本论文选择Aβ肽全长的中间段Aβ12-35作为抗原,筛选其纳米抗体,并通过E.coli表达系统制备纳米抗体,并探讨了其对Aβ聚集能力的影响。本论文研究内容如下：(1)纳米抗体基因序列的筛选。以Aβ12-35单体作为抗原,通过抗原的定点固载,利用噬菌体展示库技术,经过4轮淘选,ELISA测定亲和活性,从Aβ42噬菌体免疫库中筛选得到DP4、DP6、DP18和DP36四种纳米抗体基因序列。(2)纳米抗体的表达纯化。通过PCR扩增、双酶切等方法将4种纳米抗体基因克隆到表达载体pET23a;重组质粒在宿主菌E.coli Shuffle T7和E.coli Origami 2(DE3)中实现了可溶性表达。纯化后其纯度均能达到90%以上。经ELISA活性检测,S-DP4, O-DP4, S-DP6, O-DP6表现出较好的抗原结合活性。Biacore测定其亲和常数,S-DP6亲和常数最高,约为10-7,其他三种亲和常数在10-6,结果与ELISA基本相符。(3)纳米抗体的功能研究。ELISA结果表明,纳米抗体S-DP6除了能识别Aβ12-35单体和聚集体,还能识别Aβ40和Aβ42的单体和聚集体；体外利用ThT荧光法和电子显微镜检测发现,所获得的纳米抗体S-DP6对Aβ12-35单体的聚集和纤维化有抑制作用；对已形成的Aβ12-35纤维有解聚作用。总之,本论文筛选获得的纳米抗体对多种Aβ都有识别作用,且可以影响Aβ的聚集过程,为Aβ聚集引起的疾病机理的研究提供了很好的研究材料。
[Abstract]:Alzheimer's disease (Alzheimer's disease, AD) is a severe neurodegenerative disease, and its pathogenesis is still unknown. Many studies have shown that A 尾 and its aggregates are important factors influencing the development of AD. Therefore, the development of A-尾-agglomerate inhibitors or drugs has become one of the hot research topics. In the treatment of AD based on anti-A 尾 antibody, humanized polyclonal or monoclonal antibodies and small molecular single chain antibodies are used. Compared with these methods, nano-antibody can easily cross the blood-brain barrier because of its low preparation cost. More and more attention has been paid to antibody therapy. In addition, previous studies have shown that the nitrogen terminal of A 尾 does not participate in the aggregation of A 尾, and the antibody which can recognize the nitrogen terminal of A 尾 in clinical trials has to stop clinical trials because it can cause a stronger inflammatory reaction in the body. The A 尾 intermediate fragment is the key site associated with A 尾 aggregation and the toxic fragment. It is easy to form a 尾 folding structure, and 尾 folding is the main structural feature of A 尾 aggregation, and the inflammatory response to the antibody in A 尾 accumulation region is relatively low. Therefore, it is important to screen nano-antibodies that can specifically bind to the middle of A 尾 in order to inhibit the aggregation of A 尾 and to explore the mechanism of A 尾 aggregation. In this paper, A 尾 12-35, the intermediate part of A 尾 peptide, was selected as the antigen to screen its nanometer antibody, and the nanometer antibody was prepared by E. coli expression system, and the effect on the aggregation ability of A 尾 was discussed. The main contents of this thesis are as follows: (1) screening of nano-antibody gene sequence. Using A 尾 12-35 monomer as antigen, the affinity activity was determined by using phage display library technique and four rounds of Amoy Elisa. (2) the expression and purification of dp4, dp6, DP18 and DP36 were obtained from the A 尾 42 phage library. (2) the expression and purification of the antibody. By PCR amplification and double enzyme digestion, four kinds of nano-antibody genes were cloned into the expression vector pET23a.The recombinant plasmid was expressed in E. coli Shuffle T7 and E. coli Origami 2 (DE3). After purification, the purity was above 90%. S-DP4, O-DP4, S-DP6, O-DP6 showed good antigen-binding activity. Biacore showed that the affinity constant of S-DP6 was the highest, about 10-7, and the other three affinity constants were 10-6. The antibody S-DP6 can recognize not only A 尾 12-35 monomer and aggregates, but also A 尾 40 and A 尾 42 monomers and aggregates. The obtained nano-antibody S-DP6 can inhibit the aggregation and fibrosis of A 尾 12-35 monomer and depolymerize the formed A 尾 12-35 fiber. In conclusion, the nanometer antibodies obtained in this paper can recognize many kinds of A 尾, and can affect the aggregation process of A 尾, which provides a good material for the study of the disease mechanism caused by A 尾 aggregation.
【学位授予单位】：大连理工大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R749.16

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