基于酵母表面展示系统的HIV抗体分离和表达

发布时间：2018-03-05 19:50

本文选题：酵母展示　切入点：HIV　出处：《北京协和医学院》2013年博士论文　论文类型：学位论文

【摘要】：人类免疫缺陷病毒(Human Immunodeficiency Virus, HIV)的防治问题是当今全球临床研究人员亟需解决的重要课题。由于HIV具有高度变异的特点,研究人员在寻找能够对HIV病毒提供具有长效广谱作用的中和抗体的过程中一直面临着极大的困难。本文将通过设计有效的实验技术,分离并表达可与HIV特异性结合的抗体。为此,需要解决两个重要问题,如何选择靶抗原以及如何展示抗体文库。针对第一个问题,研究人员通过分析当前已经找到的HIV广谱中和抗体,发现这些抗体对应的病毒抗原表位主要分布在HIV包膜糖蛋白gp120/gp41上的一些保守位点。因此,本课题组研究人员提出了找到合适靶抗原的新方法,即根据病毒表面的保守表位来设计并合成抗原肽。基于前期的研究结论,本文提出将靶抗原的设计定位于HIV广谱中和抗体10E8所对应的表位,即gp41近膜区域(membrane-proximal external region, MPER)上的一段保守位点。同时根据此保守位点进行化学合成与修饰,得到与实际抗原功能相近的一段多肽,从而利用合成的肽段筛选抗体文库。解决第二个问题,需要从众多的表面展示技术中,挑选一种用来展示抗体文库。本文选择酵母表面展示系统进行抗体文库的展示。一方面,由于酵母细胞作为真核细胞,在蛋白质翻译后修饰以及哺乳动物蛋白质加工等方面具有优势,更适合对人类抗体片段的表达及展示。另一方面,在实验过程中,酵母细胞也易于培养和进行细胞筛选。同时,本文选取易于基因操作和表达的单链抗体(Single Chain Fragment Variable,scFv)文库展示在酵母细胞表面。在酵母细胞表面展示了包含有109个单链抗体的非免疫人类单链抗体文库,其中存在产生广谱中和抗体的基因。本文提出的实验技术包括如下三个步骤：首先,对酵母表面展示的单链抗体文库进行两轮免疫磁珠分选(Megnetic Activated Cell Sorting, MACS)及两轮流式细胞分选(Fluorescence Activated Cell Sorting, FACS)过程;其次,从筛选出的抗体文库中随机挑选30株进行基因测序,对得到的12株不同单克隆进行鉴定,其中有4株为阳性结果克隆；最后,将这4株阳性克隆进行单链抗体的分泌、纯化,并进一步构建载体,将其以全长形式进行表达,经再次验证,以全长形式表达的抗体仍能够与抗原肽结合。实验结果表明,该技术可以实现从酵母细胞表面展示技术展示的单链抗体文库中,分离和表达出可与靶抗原特异性结合的抗体,并建立从酵母表面展示的单链抗体文库中筛选针对靶抗原的抗体的技术平台。本文提出的实验技术具有广阔的应用前景,对于未来的疫苗研发具有重要的意义。通过该实验技术,研究人员可以不断地设计与改造抗原肽,从而找到能够从抗体文库中筛选出广谱HIV中和抗体的肽段,并将此肽段作为病毒疫苗的前身,指导后续的疫苗研发。
[Abstract]:The prevention and treatment of human Immunodeficiency virus (HIV) is an important problem for clinical researchers all over the world. The researchers have been facing great difficulties in finding neutralizing antibodies to HIV that have a long and broad spectrum. This paper will design an effective experimental technique. In order to isolate and express antibodies that can specifically bind to HIV, two important problems need to be solved: how to select the target antigen and how to display the antibody library. In response to the first question, by analyzing the HIV broad-spectrum neutralizing antibodies that have been found so far, the researchers found that the antigenic epitopes corresponding to these antibodies are mainly located at some conserved sites on the HIV envelope glycoprotein gp120/gp41. Our researchers have proposed a new method to find suitable target antigens, that is, to design and synthesize antigenic peptides according to the conserved epitopes on the surface of viruses. In this paper, we propose that the design of target antigen be located in the epitope corresponding to HIV broad-spectrum neutralizing antibody 10E8, that is, a conserved site in the membrane proximal external region of gp41, and the conserved site is chemically synthesized and modified. A segment of peptide was obtained which was similar to the actual antigen function, and the antibody library was screened by using the synthetic peptide fragment. To solve the second problem, we need to select one of the many surface display techniques to display the antibody library. In this paper, we choose the yeast surface display system to display the antibody library. It has advantages in protein post-translational modification and mammalian protein processing, and is more suitable for the expression and display of human antibody fragments. On the other hand, yeast cells are also easy to culture and screen cells during the experiment. The single Chain Fragment variable scFvvlibrary, which is easy to manipulate and express genes, was selected to display on the surface of yeast cell. The unimmunized human scFv library containing 109 scFv was displayed on yeast cell surface. There are genes that produce broad-spectrum neutralizing antibodies. The experimental techniques proposed in this paper include the following three steps: firstly, two rounds of immunomagnetic Activated Cell sorting (Macs) and two rounds flow cytometric Activated Cell sorting (FACSs) were performed on the single chain antibody library displayed on the surface of yeast. From the selected antibody library, 30 strains were randomly selected for gene sequencing, and 12 different monoclonal clones were identified, of which 4 were positive clones. Finally, the four positive clones were secreted and purified by scFv. Furthermore, the vector was constructed and expressed in full-length form. It was proved that the antibody expressed in full-length form could still bind to the antigenic peptide. This technique can be used to isolate and express antibodies that can specifically bind to target antigen from the single chain antibody library displayed by yeast cell surface display technology. A technique platform for screening antibodies against target antigens from single-chain antibody library displayed on yeast surface was established. The experimental technology proposed in this paper has a broad application prospect. It is of great significance for future vaccine development. Through this experimental technique, researchers can continuously design and modify antigenic peptides and find out the peptides that can be screened from antibody libraries to identify broad spectrum HIV neutralizing antibodies. The peptide was used as the precursor of the virus vaccine to guide the subsequent vaccine development.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R512.91

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